Autophagy-related protein 1 orchestrates autophagy initiation and feedback degradation in Alternaria alternata

Autophagy-related protein 1 orchestrates autophagy initiation and feedback degradation in Alternaria alternata | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Autophagy-related protein 1 orchestrates autophagy initiation and feedback degradation in Alternaria alternata Celine Yen Ling Choo, Hsin-Yu Lu, Kuang-Ren Chung, Pei-Ching Wu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8345089/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract Background Autophagy plays an essential role in fungal development and stress adaptation, yet its regulatory mechanisms in filamentous fungi remain incompletely understood. We functionally characterized Alternaria alternata Atg1 (AaAtg1), a serine/threonine kinase, and demonstrated its dual roles in autophagy initiation and flux modulation. Results Deletion of AaAtg1 abolishes autophagosome formation and autophagic flux, impairs peroxisome degradation, and leads to hypersensitivity to oxidative stress, as well as reduced virulence. AaAtg1 physically interacts with core autophagy proteins AaAtg13 and AaAtg8, and its vacuolar degradation is AaAtg8-dependent. Structure-guided mutagenesis of the Atg8-family interacting motif (AIM) disrupts AaAtg1–AaAtg8 binding in yeast two-hybrid assays but not in bimolecular fluorescence complementation, suggesting partial functional retention in vivo . Intriguingly, AIM mutations do not impair autophagy; instead, some transformants exhibit elevated autophagic activity, suggesting a potential negative regulatory role of AIM in autophagy tuning. Conclusions These findings reveal a noncanonical feedback mechanism in which AaAtg8 facilitates AaAtg1 degradation to modulate autophagic output. Our study elucidates the structure–function relationship of AaAtg1 and uncovers a dual regulatory mechanism that coordinates autophagy progression and stress adaptation in the plant-pathogenic fungus. AIM Peroxisome turnover Nutrient sensing Oxidative stress tolerance Fungal pathogenicity Autophagic flux regulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Eukaryotic cells utilize autophagy, an intracellular degradation and recycling process, to maintain cellular homeostasis and adapt to environmental stress [ 1 ]. Autophagy is mediated by the coordinated action of an intricate machinery involving a group of autophagy-related proteins (Atgs) [ 2 ]. The process begins at the isolation membrane, where several Atg proteins are assembled to form a pre-autophagosomal structure (PAS) (also called phagophore assembly site) [ 2 , 3 ]. A key player in this early step is Atg1, a serine/threonine kinase homologous to the mammalian Unc-51-like kinase 1 (ULK1) [ 4 , 5 ]. In yeast, Atg1 forms a complex with Atg13 and associates with the Atg17-Atg31-Atg29 trimer to localize to the PAS and initiate autophagosome formation [ 6 – 9 ]. Subsequently, PAS expands to form double-membraned autophagosomes that engulf damaged proteins and organelles [ 8 ]. This self-recycling mechanism is crucial for cell survival under stressful conditions and for maintaining cellular homeostasis under ordinary circumstances. Autophagy is required for various cellular processes in filamentous fungi, including growth, cellular differentiation, development, conidiation, appressorium formation, and virulence [ 10 – 13 ]. While the importance of autophagy in nutrient limitation and stress responses is well recognized, its regulatory mechanisms, particularly the interplay among key autophagy proteins, remain incompletely understood in plant-pathogenic fungi. Atg1 phosphorylation is regulated by several kinases, including the Target of Rapamycin kinase (TOR) [ 14 , 15 ], the AMP-activated protein kinase (AMPK) [ 16 ], and protein kinase A (PKA) [ 17 , 18 ]. Nutrient availability critically determines TOR activity, which in turn modulates Atg1 function. Under nutrient-poor conditions, TOR is suppressed, allowing Atg13 to remain dephosphorylated and to bind Atg1 with high affinity, thereby activating autophagy [ 19 ]. Conversely, under nutrient-rich conditions, TOR phosphorylates Atg13, weakening its interaction with Atg1 and inhibiting autophagy. The phosphorylated Atg1 phosphorylates Atg13 to regulate autophagy initiation and directly phosphorylates Atg11 to control selective autophagy processes such as pexophagy [ 19 – 21 ]. This dynamic phosphorylation-dephosphorylation cycle ensures precise regulation of autophagy in response to cellular nutrient status. Autophagosome membrane expansion requires the ubiquitin-like protein Atg8 [ 22 ], which conjugates to phosphatidylethanolamine (PE) and anchors to the membrane surface [ 23 ]. In mammals, Atg8 homologs, such as microtubule-associated protein light chain 3 (LC3) or the gamma-aminobutyric acid A receptor-associated protein (GABARAP) [ 24 ], also serve as markers for monitoring autophagy formation [ 24 , 25 ]. Beyond facilitating membrane expansion, the Atg8-PE conjugate plays key roles in cargo selection and autophagosome-vacuole fusion [ 26 , 27 ]. It interacts with receptor proteins containing Atg8-family-interacting motifs (AIM) in yeast or LC3-interacting region (LIR) in mammals, thereby sequestering target proteins or damaged organelles during autophagosome expansion [ 28 , 29 ]. Additionally, Atg8 recruits Atg1/ULK1 to autophagosomes, facilitating their maturation and fusion with vacuoles, a process crucial for degrading the Atg1/Atg13 complex [ 23 , 29 ]. This interaction relies on the AIM of Atg1, which is essential for its binding to Atg8, as demonstrated in yeast. Mutations in the AIM disrupt Atg1-Atg8 binding, impair Atg1 transport to vacuoles, and hinder autophagy [ 27 , 30 ]. Alternaria alternata is a necrotrophic fungal pathogen that causes diseases in more than 100 plant species and obtains nutrients exclusively from dead tissues [ 31 ]. Many A. alternata pathogens produce host-selective toxins, which are major contributors to pathogenicity [ 32 ]. During host colonization, A. alternata must also overcome the toxicity of reactive oxygen species (ROS) generated by the plant. We have previously shown that pexophagy, the selective autophagic degradation of peroxisomes, plays a crucial role in ROS resistance and cellular homeostasis in A. alternata [ 33 , 34 ]. Moreover, our recent study revealed that autophagy regulates siderophore production and intracellular iron homeostasis, thereby contributing to ROS detoxification; notably, Δ AaAtg1 failed to produce siderophores and displayed hypersensitivity to the iron chelator bathophenanthrolinedisulfonic acid (BPS) [ 35 ]. In this study, we sought further to dissect the role of AaAtg1 in stress-induced autophagy, focusing on its contributions to autophagic flux and broader regulatory functions. Our results indicate that AaAtg1 is essential for autophagy and that its degradation depends on interaction with AaAtg8. Intriguingly, mutations in the AIM of AaAtg1 disrupted its interaction with AaAtg8 but paradoxically enhanced autophagic activity in certain mutant strains. These observations point to a previously unrecognized dual role of AaAtg1 in both initiating and modulating autophagy, highlighting the complexity of autophagy regulation in filamentous fungi. Results AaAtg1 is required for growth and development To investigate the role of AaAtg1 in autophagy and pathogenesis in A. alternata , two deletion mutant strains (designated Δ AaAtg1 -D6 and Δ AaAtg1 -D7) were generated using a split-marker approach. Compared with the wild type, Δ AaAtg1 exhibited slower growth, with 10% and 30% reductions in potato dextrose agar (PDA) and minimal medium (MM), respectively (Supplementary Figure S1 A). Wild-type conidia were septated and ellipsoidal or cylindrical, whereas Δ AaAtg1 produced slender conidia and abundant hyphal fragments (Supplementary Figure S1 B, C). Germination of Δ AaAtg1 conidia was markedly slower than that of the wild type. Additionally, Δ AaAtg1 produced significantly fewer conidia and appressorium-like structures than the wild type (Supplementary Figure S1 D, E). Genetic complementation of Δ AaAtg1 with a functional AaAtg1 copy (Cp8 strain) restored wild-type growth, conidiation, appressorium formation, and germination. AaAtg1 is required for autophagy Transmission electron microscopy (TEM) examination revealed that autophagic vacuoles (AVs) containing dense dark contents were present in the wild-type hyphae grown in MM-N (minimal medium without nitrogen) (Fig. 1 A). In contrast, Δ AaAtg1 lacked such AVs; most of them contained no dense contents. Western blot analyses revealed that Δ AaAtg1 expressing GFP-AaAtg8 failed to initiate autophagy when exposed to hydrogen peroxide (H 2 O 2 ) or MM-N, while the wild type accumulated free GFP, indicative of active autophagy (Fig. 1 B). Fluorescence microscopy further confirmed these findings. In wild-type hyphae incubated in MM-N or treated with H 2 O 2 for 24 h, round blue fluorescent spots [7-amino-4-chloromethylcoumarin (CMAC)-stained vacuoles] were colocalized with green fluorescent spots from GFP-AaAtg8, indicating autophagosomes within vacuoles (Fig. 2 ). In contrast, in Δ AaAtg1 hyphae cultured in MM-N, green fluorescent spots were observed near vacuoles but did not colocalize with them. When exposed to H₂O₂ for 24 h, Δ AaAtg1 hyphae exhibited blurry green fluorescence, weak CMAC staining, and abnormal hyphal morphology, suggesting cell death. AaAtg1 is involved in peroxisome turnover To determine the role of AaAtg1 in pexophagy, a mCherry tag fused with serine-lysine-leucine (SKL) tripeptides was expressed in wild-type and Δ AaAtg1 strains to label peroxisomes. Fluorescence microscopy revealed that in both WT/mCherry-SKL and Δ AaAtg1 /mCherry-SKL strains, red fluorescence (representing peroxisomes) was not localized in vacuoles when cultured in potato dextrose broth (PDB) for 24 h. In the wild-type hyphae, after being shifted to MM-N or treated with H 2 O 2 , red fluorescence was observed in the vacuoles, indicating active pexophagy (Fig. 3 ). In contrast, red fluorescent spots were not localized in the vacuoles of Δ AaAtg1 /mCherry-SKL hyphae cultured in MM-N, suggesting impaired pexophagy. Furthermore, under H₂O₂ treatment, Δ AaAtg1 /mCherry-SKL hyphae exhibited deformed morphology, lacked distinct vacuoles, and showed no discernible red fluorescence, indicating severe cellular damage and disrupted peroxisomal turnover. AaAtg1 is involved in the detoxification of ROS Due to the observed shrinkage and accumulation of dense materials in Δ AaAtg1 hyphae exposed to H 2 O 2 , sensitivity tests were conducted on PDA amended with H 2 O 2 , tert -butyl hydroperoxide (tBHP), or cumyl peroxide. Compared to the wild type, Δ AaAtg1 exhibited increased hypersensitivity to all three oxidants (Supplementary Figure S2 A, B). However, Δ AaAtg1 displayed wild-type sensitivity to single oxygen-producing compounds (rose Bengal and eosin Y) and cell wall stressors, sodium dodecyl sulfate (SDS), Congo red, and calcofluor white (data not shown). When treated with H 2 O 2 and stained with 2'-7'-dichlorofluorescein diacetate (DCFHDA), Δ AaAtg1 hyphae showed stronger green fluorescence, indicating greater ROS accumulation than the wild type (Supplementary Figure S2 C). Similarly, propidium iodine (PI) staining revealed significantly stronger red fluorescence in Δ AaAtg1 hyphae, indicating a higher proportion of dead cells after H₂O₂ treatment. AaAtg1 is required for full virulence but not for host-selective toxin production Pathogenicity tests conducted on detached calamondin leaves revealed a significant reduction of necrotic lesions after inoculation with conidial suspensions of Δ AaAtg1 4 days post-inoculation (dpi) (Supplementary Figure S3A). In contrast, leaves inoculated with conidia from wild-type and CP8 strains developed necrotic lesions within the same period. Pre-wounding of leaves did not enhance lesion formation by the Δ AaAtg1 strain. Microscopic examination revealed that conidia from wild-type and CP8 strains germinated rapidly and formed hyphae on leaf surfaces at 1 dpi (Supplementary Figure S3B). However, conidia from Δ AaAtg1 showed delayed germination on leaves until 2–3 dpi (data not shown), correlating with the delayed necrotic lesion development. Thin-layer chromatography (TLC) and High-performance liquid chromatography (HPLC) analyses revealed no quantitative difference in host-selective ACT toxin production between wild-type and Δ AaAtg1 strains (Supplementary Figure S4A, B). AaAtg1 physically interacts with both AaAtg13 and AaAtg8 in A. alternata To determine whether AaAtg1 physically interacts with other core autophagy components, we performed Yeast two-hybrid (Y2H) assays using AaAtg13 and AaAtg8 as potential binding partners. Yeast cells expressing AaAtg1 fused to the GAL4 activation domain (AaAtg1-AD) and either AaAtg13 or AaAtg8 fused to the binding domain (AaAtg13-BD, AaAtg8-BD) thrived on QDO/X/A selective medium (Fig. 4 A, B), indicating direct interactions between AaAtg1 and both proteins. These interactions were further validated in vivo using bimolecular fluorescence complementation (BiFC) assays. Co-expression of GFP N -AaAtg13 with GFP C -AaAtg1, or GFP N -AaAtg8 with GFP C -AaAtg1, in A. alternata protoplasts led to the appearance of green fluorescent signals along the hyphal compartments (Fig. 4 C, D), while the negative control strains expressing only split GFP fragments showed no fluorescence, confirming the specificity of these interactions. To further confirm the interaction between AaAtg1 and AaAtg8 under native conditions, co-immunoprecipitation (Co-IP) was performed. Cell lysates from A. alternata strains co-expressing HA-tagged AaAtg1 and GFP-tagged AaAtg8 were immunoprecipitated using anti-GFP antibodies. Western blotting detected HA-AaAtg1 in the precipitated complex, confirming its association with GFP-AaAtg8 in vivo (Fig. 4 E). Together, AaAtg1 physically interacted with both AaAtg13 and AaAtg8, supporting its central role as a scaffold protein coordinating autophagy-related protein assembly in A. alternata . The AIM region affects the AaAtg1 structure and its interaction with AaAtg8 Amino acid sequence analysis revealed that AaAtg1 possesses several conserved domains associated with autophagy regulation. At the N-terminus, AaAtg1 contains a canonical serine/threonine kinase domain. In contrast, its C-terminal region harbors two microtubule-interacting and transport (MIT) domains, which are predicted to mediate interaction with AaAtg13 (Supplementary Figure S5A). Additionally, sequence inspection identified a putative AIM/LIR, characterized by the conserved sequence EGFVFVEK, located between the kinase and MIT domains (Supplementary Figure S5B). This motif could likely serve as a docking site for AaAtg8 and is conserved among Atg1 orthologs in other filamentous fungi. To evaluate the structural impact of the AIM region in AaAtg1, we used AlphaFold2 to model two variants: a deletion mutant (ΔFVFV; residues 532–535 removed) and a point-mutated version (AAVFAA; E GFVFVE K → E AAVFAA K). Superimposition with the wild-type model revealed that the ΔFVFV deletion induced a substantial global structural rearrangement (RMSD = 30.290) (Fig. 5 A). In contrast, the AAVFAA substitution caused only minor overall changes (RMSD = 2.158) (Fig. 6 A). Nonetheless, both mutants exhibited pronounced local alterations at the AIM region, notably affecting loop structure and side-chain positioning (Figs. 5 B and 6 B). To investigate whether these structural changes disrupt protein–protein interactions, we performed Y2H and BiFC assays. In Y2H, neither the ΔFVFV nor AAVFAA variants showed detectable interaction with AaAtg8 (Fig. 7 A, B), suggesting reduced binding affinity in vitro . However, BiFC assays revealed green fluorescence in both mutant strains (Fig. 7 C, D), indicating residual or altered interaction in vivo . The AIM mutation could increase autophagy under certain conditions To investigate the role of the AIM in autophagy flux, Δ AaAtg1 co-expressing AaAtg1 ΔFVFV (deletion) or AaAtg1 AAVFAA (point mutation) with GFP-AaAtg8 were further examined. The control strain, Δ AaAtg1 co-expressing wild-type AaAtg1 and GFP-AaAtg8, displayed uniform green fluorescence in hyphae grown in PDB and distinct fluorescent spots after being shifted to MM-N (Fig. 8 A). Δ AaAtg1 /AaAtg1 ΔFVFV transformants exhibited two distinct fluorescent patterns, approximately in a 1:1 ratio. One group (14 transformants), exemplified by transformant No. 14, exhibited green fluorescent patterns and intensities similar to those of the control strain. The other group (18 transformants), represented by transformant No. 18, displayed weak fluorescent spots under MM-N. Western blot analysis revealed that free GFP cleavage from GFP-AaAtg8 was 51% in transformant No. 14, comparable to the control strain (67%) (Fig. 8 B). However, transformant No. 18 exhibited nearly 90% of free GFP under MM-N conditions, indicating enhanced autophagic flux. Similarly, Δ AaAtg1 /AaAtg1 AAVFAA transformants also showed two distinct fluorescent patterns. Transformant No. 10 displayed strong fluorescence comparable to the control strain, while other transformant No. 12 exhibited weak fluorescence (data not shown). Western blot analysis confirmed ~ 50% free GFP in transformant No. 10, similar to the control, but ~ 97% free GFP in transformant No. 12, further supporting enhanced autophagy when the AIM region was mutated (Fig. 8 C). Localization of AaAtg1 to vacuoles is triggered by nitrogen starvation and mediated by AaAtg8 To investigate the localization of AaAtg1 during autophagy and the role of AaAtg8 in this process, GFP-AaAtg1 was expressed in Δ AaAtg1 and Δ AaAtg8 strains, and the resulting transformants were examined microscopically (Fig. 9 ). In Δ AaAtg1 /GFP-AaAtg1 hyphae (complementation strain), green fluorescence was uniform after being shifted to PDB for 6 h, with no visible vacuoles. Following 6 h in MM-N, vacuoles became visible, but green fluorescence was barely detectable within them. After 24 h in PDB or MM-N, green fluorescence formed distinct patches, suggesting vacuolar localization during prolonged starvation. In contrast, Δ AaAtg8 /GFP-AaAtg1 hyphae emitted uniform green fluorescence with no visible vacuoles after 6 h in PDB. At 24 h, vacuoles became visible, but green fluorescence was excluded from vacuoles. Similarly, after 6 or 24 h in MM-N, vacuoles in Δ AaAtg8 /GFP-AaAtg1 hyphae contained no green fluorescence, indicating the requirement of AaAtg8 for the proper localization of AaAtg1 to vacuoles during autophagy. Discussion The necrotrophic pathogen A. alternata produces host-selective toxins [ 36 ] and cell wall-degrading enzymes [ 37 ] to kill its host plant, leading to the accumulation of toxic ROS [ 38 ]. A. alternata must detoxify ROS to obtain nutrients from dead cells [ 39 ]. Current studies have shown the important role of AaAtg1 in ROS- and starvation-sensing, regulating autophagy initiation and cargo degradation. AaAtg1 is required for normal autophagy formation, which plays a crucial role in growth, conidiation, conidial germination, appressorial development, and pathogenesis. AaAtg1 is also responsible for autophagy-mediated peroxisome turnover. AaAtg1-mediated autophagy formation plays an important role in cellular resistance to ROS and cell survival under oxidative stress and starvation. AaAtg1 physically interacts with AaAtg8 and AaAtg13 to initiate autophagy, and AaAtg8 is required for AaAtg1 degradation in the vacuoles. We have further demonstrated that the AIM region of AaAtg1 is required for binding to AaAtg8 in Y2H but not in BiFC assays. Surprisingly, mutating the AIM region did not affect autophagy formation in some transformants but did in others. This suggests a leaky regulation in autophagy formation once the AIM region is mutated. In both yeast and mammals, the Atg1/ULK1 complex integrates nutrient starvation signals and recruits core autophagy proteins to the PAS to initiate autophagy [ 5 ]. However, in A. alternata , deletion of AaAtg1 does not abolish PAS-like structures. As shown in Fig. 2 , GFP-AaAtg8 puncta remain detectable under nutrient-rich conditions, appearing as small bright fluorescent dots, suggesting that AaAtg1 is not essential for the basal recruitment of Atg8 to the PAS. Under nitrogen starvation, larger GFP-AaAtg8 puncta accumulate adjacent to vacuoles in the Δ AaAtg1 mutant but fail to enter them, and show no GFP cleavage. These results indicate a complete block in autophagic flux once AaATg1 is impaired. This phenotype partially resembles that of Saccharomyces cerevisiae , in which Atg1 is dispensable for the initial PAS localization of Atg8 and Atg9 but is required for autophagosome formation and progression of autophagy [ 6 , 40 ]. In A. alternata , however, AaAtg1 appears to play a more stringent role. Although PAS-like puncta can form without it, subsequent steps, including autophagosome maturation, vacuolar fusion, and cargo degradation, are completely impaired. This strict dependency is corroborated by TEM and proteolytic assays, which confirm the absence of autophagic vacuoles in Δ AaAtg1 under both nitrogen starvation and oxidative stress. Δ AaAtg1 is highly sensitive to H₂O₂ and other peroxides. Although PAS and autophagosomes are present in Δ AaAtg1 hyphae under nutrient-poor conditions (e.g., MM-N), these structures are absent following H₂O₂ treatment, likely due to ROS-induced cell death. In contrast, wild-type hyphae form autophagic vacuoles under both nitrogen starvation and oxidative stress. DCFHDA staining reveals elevated intracellular H₂O₂ accumulation in Δ AaAtg1 compared to the wild type, and PI staining confirms extensive cell death after H₂O₂ exposure. These findings underscore the importance of autophagy in maintaining redox homeostasis and promoting survival under oxidative stress. Previous studies show that H₂O₂ enhances peroxisome turnover in A. alternata , leading to decreased peroxisome levels [ 34 ]. We therefore investigated whether AaAtg1-mediated autophagy contributes to peroxisome degradation. Both nitrogen starvation and H₂O₂ induce peroxisome aggregation and translocation into vacuoles in the wild type (Fig. 3 ). In Δ AaAtg1 , however, peroxisomes fail to undergo these changes: under MM-N, they remain scattered outside vacuoles, and after H₂O₂ exposure, they become undetectable. Given that peroxisomes are significant sites of H₂O₂ production [ 41 ], and defects in peroxisome biogenesis render fungal cells ROS-sensitive [ 42 ], the failure to degrade peroxisomes likely contributes to ROS accumulation and cell death in Δ AaAtg1 . Together, these results highlight the essential role of AaAtg1 in pexophagy and oxidative stress resistance. In yeast, Atg1 interacts with Atg8 in an AIM-dependent manner [ 27 , 30 ]. The AIM region (EGFVFVEK) of AaAtg1 serves as a critical docking site for AaAtg8, as demonstrated by Y2H and BiFC assays. Y2H assays show that AIM mutations (ΔFVFV or EAAVFAAK) disrupt AaAtg8 binding, whereas BiFC assays reveal residual in vivo interaction, suggesting that the cellular context may enable weak or transient interactions. This points to a subtle regulatory mechanism of autophagy in A. alternata . Unexpectedly, AIM mutations do not uniformly suppress autophagy. Compared to wild-type, some transformants exhibit increased autophagic flux, as evidenced by stronger GFP-AaAtg8 cleavage. These findings imply that impaired AaAtg1–AaAtg8 interaction may reduce AaAtg1 degradation, leading to kinase stabilization and excessive autophagy activation. This "leaky regulation" contrasts sharply with yeast, where AIM mutations consistently suppress autophagy [ 30 ]. Together, our data suggest that the AIM region in A. alternata serves not only as a docking site but also as a gatekeeper that modulates autophagic intensity, highlighting a distinct regulatory mechanism that prevents detrimental hyperactivation. Localization studies reveal that AaAtg1 translocates to vacuoles under nitrogen starvation, a process that is dependent on AaAtg8. In Δ AaAtg8 hyphae, GFP-AaAtg1 fails to accumulate in vacuoles, indicating that AaAtg8 mediates the vacuolar sequestration and degradation of AaAtg1 during autophagy. This AaAtg8-dependent turnover likely functions as a feedback mechanism that downregulates autophagic activity and maintains homeostasis during prolonged stress. This observation aligns with findings in S. cerevisiae , where Atg1 undergoes degradation via an AIM/LIR–dependent mechanism [ 23 ]. Autophagy deficiency in Δ AaAtg1 correlated with marked virulence attenuation, likely due to combined effects on hyphal growth, sporulation, and ROS detoxification. In contrast, AIM mutations did not impair virulence (data not shown), yet they revealed a possible inhibitory role of the AIM-mediated interaction in restricting excessive autophagy. This suggests that the AIM region not only facilitates Atg8 binding but also serves as a checkpoint to prevent overactivation of the autophagic pathway. Conclusion This study demonstrates that AaAtg1 functions not only as a trigger for autophagy initiation but also as a substrate of autophagic degradation, thus constituting a negative feedback loop, a concept rarely described in plant-pathogenic fungi. The paradoxical enhancement of autophagic flux in AIM mutants may reflect the loss of this regulatory brake, a mechanism warranting further investigation. Together, these studies highlight AaAtg1 as a macromolecular scaffold that coordinates protein–protein interactions, signal transduction, and autophagic flux regulation in A. alternata . These insights expand our understanding of fungal stress adaptation and virulence control via autophagy, offering new perspectives on the molecular mechanisms that govern autophagy homeostasis in filamentous plant pathogens. Materials and Methods Fungal strains, conidiation, germination, and sensitivity assays The wild-type strain of A. alternata used for transformation and mutagenesis has been previously characterized [ 43 ]. Fungal strains were point-inoculated onto PDA (Difco, Franklin Lakes, NJ, U.S.A.) or MM [ 44 ] agar using a sterile toothpick and incubated at 28°C. For conidiation, fungal strains were grown on PDA without parafilm sealing under light for 5 days. Conidia were collected in sterile water, and germination was assessed by incubating conidia on glass slides or 96-well microtiter plates at 28°C for 6 h. Fungal sensitivity assays were conducted on PDA plates (90 mm × 15 mm Petri dishes) containing test compounds, including calcofluor white (200 µM in dimethyl sulfoxide), Congo red (75 µM in ethanol), sodium dodecyl sulfate (SDS, 0.01%), rose Bengal (30 µM), eosin Y (100 µM), hydroperoxide (H₂O₂, 20 mM), tert -butyl hydroperoxide (tBHP, 3.75 mM), and cumyl hydroperoxide (3 mM in ethanol). Unless otherwise specified, all compounds were dissolved in water. Each treatment was performed in triplicate, and experiments were independently repeated three times. Genetic modification in A. alternata The AaAtg1 sequence (accession number: KAH8628380) was retrieved from the complete genome of the A. alternata EV-MIL-31 strain. Functional motifs within AaAtg1 were identified using the InterPro database and protein domain analysis tools [ 45 ]. A multiple sequence alignment illustrating the functional domains of the Atg1 homologs across different species was generated using the Illustrator for Biological Sequences (IBS) software [ 46 ]. All Atg1 protein sequences were obtained from the National Center for Biotechnology Information (NCBI) database ( http://www.ncbi.nlm.nih.gov ). The AIM/LIR region was predicted using the iLIR server ( https://ilir.warwick.ac.uk/ ) [ 28 ]. Structure models of full-length AaAtg1 and its AIM-mutated variants were constructed using AlphaFold2 ( https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb ) [ 47 ]. Two Δ AaAtg1 mutants (D6 and D7) and a complementation strain (CP8) were previously generated and characterized [ 35 ]. Δ AaAtg1 mutants were transformed with pCB1532-GFP-AaAtg8 or pCB1532-mCherry-SKL to generate Δ AaAtg1 /GFP-AaAtg8 and Δ AaAtg1 /mCherry-SKL strains, respectively, using protoplasts from the Δ AaAtg1 D6 mutant. Protoplast preparation and fungal transformations were conducted as described previously [ 48 ]. Wild-type strains expressing GFP-AaAtg8 (WT/GFP-AaAtg8) or mCherry-SKL (WT/mCherry-SKL) from previous studies [ 33 , 34 ] were used as controls. To investigate the localization of AaAtg1 in the absence of AaAtg8, the pNEOGPE1-GFP-AaAtg1 plasmid was constructed and transformed into protoplasts of both Δ AaAtg1 and Δ AaAtg8 strains, with the latter generated in a previous study [ 34 ]. Oligonucleotide primers used in this study are listed in Supplementary Table S1 , and all plasmids are detailed in Supplementary Table S2 . Site-directed mutagenesis and plasmid construction Fungal strains were cultured in a liquid complete medium [ 49 ] on a shaker at 28°C for 2 days. Mycelium was harvested and ground in liquid nitrogen for RNA isolation using TRI-reagent (Sigma-Aldrich, St. Louis, MO, U.S.A.) and the PureLink RNA Kit (Invitrogen, Waltham, MA, U.S.A.) according to the manufacturer’s protocols. Complementary DNA (cDNA) was synthesized from the isolated RNA using iScript Reverse Transcriptase (Bio-Rad, Hercules, CA, U.S.A.). Two-step fusion PCR mutagenesis was performed using cDNA as a template to generate the AaAtg1 AIM deletion (AaAtg1 ΔFVFV ) and point mutation (AaAtg1 AAVFAA ) variants. In the first step, two overlapping cDNA fragments were amplified using specific primers (Supplementary Table S1 ). These fragments were then used as templates for a second round of PCR to generate the full-length AaAtg1 ΔFVFV or AaAtg1 AAVFAA fragment. PCR products were digested with EcoRV and KpnI and cloned into the pCB1532 vector to yield pCB1532-AaAtg1 ΔFVFV or pCB1532-AaAtg1 AAVFAA . The identity of the cloned cDNA fragments was confirmed by sequencing. Plasmids were individually transformed into protoplasts prepared from the Δ AaAtg1 D6 mutant strain. The resulting strains were used for AaAtg8-AaAtg1 AIM binding studies. To assess the effect of AIM mutations on GFP-AaAtg8 expression and autophagy formation, a pBARS-GFP-AaAtg8 plasmid containing a bialaphos resistance gene was transformed into protoplasts prepared from the Δ AaAtg1 /AaAtg1 ΔFVFV or Δ AaAtg1 /AaAtg1 AAVFAA strain. Fungal transformants were recovered on regeneration media supplemented with the appropriate selective agents, including bialaphos (400 µg/ml, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), geneticin (G418, 300 µg/mL, Amresco, Solon, OH, U.S.A.), or sulfonylurea (10 µg/ml, ChemService Inc., West Chester, PA, U.S.A.). Fungal strains were further screened and validated by PCR using specific primers (Supplementary Table S1 ). Western blot analysis Western blot analysis was performed as previously described [ 34 ]. Briefly, fungal strains were cultured in potato dextrose broth (PDB, Difco) for 24 h, then transferred to PDB, MM-N (MM without nitrogen), or 15 mM H₂O₂, and incubated for 4 h. Total proteins were extracted from mycelium using RIPA cell lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and 1% protease inhibitor cocktail (Biokit, Toufen City, Miaoli, Taiwan). Proteins were boiled in a loading buffer, separated by SDS-PAGE, and transferred onto PVDF membranes. Membranes were stained with Ponceau S to verify the transfer, blocked with Towbin’s (TSW) buffer containing 25 mM Tris, 192 mM Glycine, and often 20% methanol and 0.1% SDS, and incubated overnight at 4°C with a rabbit anti-GFP antibody (1:5000). After washing, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000, Jackson ImmunoResearch, West Grove, PA, U.S.A.). Signals were detected using LumiFlash Prime Chemiluminescent Substrate, HRP (Visual Protein, Taipei, Taiwan). Chemiluminescence images were captured with the ChemiDoc MP imaging system (Bio-Rad) and analyzed using ImageLab software (version 6.1.0, Bio-Rad). Band intensity was quantified using ImageJ software (US National Institutes of Health, Bethesda, MD, U.S.A.) ( https://imagej.net/ij/ ). Co-immunoprecipitation (Co-IP) For Co-IP experiments, an AaAtg1 fragment was amplified using primers ATG1 F EcoRV and ATG1 R KpnI, digested with EcoRV and KpnI, and cloned into the pNEOGPE1-HA plasmid containing a linker HA-tag sequence (5’-atgggcagctacccatacgatgttccagattacgct-3’) to generate pNEOGPE1-HA-AaAtg1. The plasmids pNEOGPE1-HA-AaAtg1 and pCB1532-GFP-AaAtg8 were individually or co-transformed into wild-type protoplasts, resulting in three distinct fungal strains. Fungal strains were cultured in PDB for 24 h, and total proteins were extracted using RIPA buffer amended with Ser/Thr phosphatase inhibitors (1.0 mM Na 3 VO 4 and 1.0 mM/l NaF; Sigma-Aldrich). Immunoprecipitation was performed using GFP-Trap Agarose beads (Chromotek, Islandia, NY, U.S.A.) according to the manufacturer’s instructions. Proteins were eluted from magnetic beads using 2× Laemmli buffer, and 25 µl of the sample was subjected to western blot analysis. Actin served as the internal control. Yeast two-hybrid (Y2H) assays Y2H assays were conducted using the GAL4-based Matchmaker Yeast Two-Hybrid System (Clontech Laboratories, Mountain View, CA, U.S.A.). Full-length coding regions of AaAtg1, AaAtg1 ΔFVFV , and AaAtg1 AAVFAA were individually amplified, fused with GAL4 activation domain (AD), and cloned into the pGADT7 plasmid to generate preys (AaAtg1-AD, AaAtg1 ΔFVFV -AD, and AaAtg1 AAVFAA -AD). Similarly, the full-length coding regions of AaAtg8 and AaAtg13 were fused to the GAL4 DNA-binding domain (DBD) and cloned into the pGBKT7 vector as baits (AaAtg8-BD and AaAtg13-BD). Plasmids were propagated in Escherichia coli and introduced into the yeast Y187 or Y2HGold strains according to the manufacturer’s protocol (Clontech). Negative (T-prey x Lam-bait) and positive (T-prey x p53-bait) controls were included to validate the assay. Four independent transformants for each experimental pairing were tested. Yeast strains were plated on selective SD/–Leu/–Trp/–Ade/–His agar plates containing X-α-Gal (40 µg/ml) and Aureobasidin A (200 ng/ml) (QDO/X/A). Growth on the selective medium indicated a strong interaction between the tested proteins. Primers used for plasmid construction are listed in Supplementary Table S1 , with restriction enzyme recognition sites incorporated to facilitate cloning. Bimolecular fluorescence complementation (BiFC) assay The N-terminal half of GFP (GFP N ) was amplified from pCB1532-GFP-AaAtg8 by PCR and subcloned into pHygroGPE1 to yield pHygroGPE1-GFP N . The C-terminal half of GFP (GFP c ) was cloned into pNEOGPE1 to result in pNEOGPE1-GFP C . Full-length coding sequences of AaAtg1, AaAtg1 ΔFVFV , and AaAtg1 AAVFAA were individually inserted into the pNEOGPE1-GFP C vector to generate three plasmid constructs (Supplementary Table S2 ). Similarly, AaAtg8 and AaAtg13 coding sequences were individually cloned into pHygroGPE1-GFP N . The resulting plasmid pairs were co-transformed into wild-type protoplasts, and transformants were recovered on regeneration medium plates supplemented with 200 µg/ml hygromycin and 300 µg/ml geneticin. Transformants were confirmed by PCR. After incubation on PDA for 3 days, hyphae were examined microscopically for green fluorescence to evaluate GFP reconstitution. Toxin production and fungal virulence Fungal strains were cultured in modified Richard's medium [ 36 ] for 21 days, and culture filtrates were collected for ACT purification using Amberlite XAD-2 resin (Sigma-Aldrich). ACT was dissolved in methanol and analyzed by TLC and HPLC, as described previously [ 50 ]. Fungal pathogenicity was evaluated by applying 10 µl of conidial suspensions (1×10⁵ cells/ml) onto detached calamondin ( Citrofortunella mitis ) leaves, either unwounded or wounded with a fine needle. Leaves treated with sterile water served as a mock control. Treated leaves were incubated in a plastic box for 3–4 days to observe necrotic lesion development. Each fungal strain was tested on at least four leaves, and the experiments were repeated 3 times. Microscopy Hyphae were stained with lactophenol-cotton blue (Sigma-Aldrich) in ethanol (4:1, v/v) for 20 min and observed using a Nikon Optiphot 2 Microscope (Tokyo, Japan). CMAC (7-amino-4-chloromethylcoumarin, 100 µM; Thermo Fisher Scientific, Waltham, MA, U.S.A.) was used to stain vacuoles. DCFHDA (2'-7'-dichlorofluorescein diacetate, Sigma-Aldrich) was used to detect cellular ROS. Cell viability was assessed using propidium iodide (PI, Sigma-Aldrich) staining. Fluorescence was detected using a ZOE Fluorescent Cell Imager (Bio-Rad) with appropriate excitation and emission wavelengths, as previously described [ 34 , 42 ]. TEM was performed as described [ 42 ]. All experiments were repeated at least twice. Statistical analysis Statistical analyses were performed using SPSS Statistics 20 software (IBM, NY, U.S.A.). Data are presented as means ± standard deviation. The homogeneity of variance was assessed with Levene’s test. Statistical significance was evaluated using either an independent sample t -test or one-way ANOVA, followed by Tukey’s honest significant difference (HSD) post hoc test ( p < 0.05). Each treatment contained at least three replicates unless otherwise specified. Abbreviations AaAtg Alternaria alternata autophagy-related protein AIM Atg8-family-interacting motif ROS Reactive oxygen species PDB Potato dextrose broth MM-N Minimal medium without nitrogen H 2 O 2 Hydrogen peroxide ACT Alternaria citri tangerine toxin Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors. Competing interests The authors have declared that no competing interests exist. Funding This work was supported by the National Science and Technology Council, Taiwan [grant numbers 112-2313-B-005-033 to K.-R. Chung, and 110-2326-B-005-001-MY3 to P.-C. Wu and K.-R. Chung]; intramural funding from China Medical University (project number CMU113-N-09 to P.-C. Wu); and the Ministry of Education, Taiwan. Authors' contributions CYLC and HYL performed the experiments, analyzed the data, validated the results, and reviewed and edited the manuscript. KRC and PCW designed the experiments, sought funding, provided supervision, validated the results, and wrote the original draft. All authors read and approved the final manuscript. Acknowledgements Not applicable References Lahiri V, Hawkins WD, Klionsky DJ. Watch what you (self-) eat: autophagic mechanisms that modulate metabolism. Cell Metab. 2019;29(4):803-26. doi: 10.1016/j.cmet.2019.03.003. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 2011;27(1):107-32. doi: 10.1146/annurev-cellbio-092910-154005. Klionsky DJ, Ohsumi Y. Vacuolar import of proteins and organelles from the cytoplasm. 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Supplementary Files Supplementarymaterial.docx SupplementaryTablesS1andS2.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 19 Mar, 2026 Reviews received at journal 18 Mar, 2026 Reviews received at journal 12 Feb, 2026 Reviewers agreed at journal 11 Feb, 2026 Reviewers agreed at journal 07 Feb, 2026 Reviewers agreed at journal 06 Feb, 2026 Reviewers invited by journal 06 Feb, 2026 Editor assigned by journal 25 Dec, 2025 Editor invited by journal 16 Dec, 2025 Submission checks completed at journal 16 Dec, 2025 First submitted to journal 15 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8345089","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588041022,"identity":"6ddde415-f6a4-425d-9a96-afa1a3738f86","order_by":0,"name":"Celine Yen Ling Choo","email":"","orcid":"","institution":"National Chung Hsing University","correspondingAuthor":false,"prefix":"","firstName":"Celine","middleName":"Yen Ling","lastName":"Choo","suffix":""},{"id":588041023,"identity":"c8ada228-41ca-45f8-8999-8439ce05b6e0","order_by":1,"name":"Hsin-Yu Lu","email":"","orcid":"","institution":"National Chung Hsing University","correspondingAuthor":false,"prefix":"","firstName":"Hsin-Yu","middleName":"","lastName":"Lu","suffix":""},{"id":588041024,"identity":"69b53fab-d6ab-4f9e-a5f0-efde64278aed","order_by":2,"name":"Kuang-Ren Chung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBACgwMMbCBajoGBB0QzE9Zi2ADRYozQwkZAizFUSWID0VrMpA8/e8y7ozZ9O/vZYxIMFdaJDfI9Bni12PClmRvznjmeu7MnL02C4Ux6YgMbDwEtPDxs0rxtx3I33OAxk2BsOwzUwrsBv8OgWtINwFr+EaHFGKKlJgGipYEILYY9bGaSc9sOGG44k2NskXAs3biNLf8DXi0GZ5ifSbxtq5M3OH7G8MaHGmvZfuZjCXi1QMFhCAVSSyhaYKCOSHWjYBSMglEwIgEArjg9payCGmUAAAAASUVORK5CYII=","orcid":"","institution":"National Chung Hsing University","correspondingAuthor":true,"prefix":"","firstName":"Kuang-Ren","middleName":"","lastName":"Chung","suffix":""},{"id":588041025,"identity":"5a84244b-0514-4528-8c99-0240a77dab04","order_by":3,"name":"Pei-Ching Wu","email":"","orcid":"","institution":"China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Pei-Ching","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2025-12-12 11:08:45","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8345089/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8345089/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102406771,"identity":"ce86bf7c-3bb2-4372-b695-9262fe3014fb","added_by":"auto","created_at":"2026-02-11 11:20:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":467957,"visible":true,"origin":"","legend":"\u003cp\u003eAaAtg1 is essential for autophagy. (\u003cstrong\u003eA\u003c/strong\u003e) TEM images of wild-type (WT) and Δ\u003cem\u003eAaAtg1\u003c/em\u003e hyphae showing autophagic vacuoles (AV), lipid bodies (L), and vacuoles (V). (\u003cstrong\u003eB\u003c/strong\u003e) Western blot of GFP-AaAtg8 using anti-GFP antibody. Fungal strains were grown in PDB for 24 h and transferred to PDB, nitrogen-starvation medium (MM-N), or 15 mM H₂O₂ for 4 h. Autophagy activity was assessed by GFP-AaAtg8 cleavage. The percentage of free GFP was calculated from band intensities. Ponceau S staining confirms equal loading. Δ\u003cem\u003eAaAtg1\u003c/em\u003e/GFP-AaAtg8 grown in PDB (Lane 1), MM-N (Lane 2), and H₂O₂ (Lane 3); WT/GFP-AaAtg8 grown in PDB (Lane 4), MM-N (Lane 5), and H₂O₂ (Lane 6); and protein marker (Lane 7).\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/158b2af27d6f633c30958ec8.png"},{"id":102406755,"identity":"df0bf683-475f-4f08-9751-352fcf37552b","added_by":"auto","created_at":"2026-02-11 11:20:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":802840,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eAaAtg1\u003c/em\u003e deficiency blocks the GFP-AaAtg8 transport into vacuoles.\u003cstrong\u003e \u003c/strong\u003eMicroscopic observation of GFP-AaAtg8\u003cstrong\u003e \u003c/strong\u003elocalization in the hyphae from the wild-type strain (WT/GFP-AaAtg8) and the \u003cem\u003eAaAtg1\u003c/em\u003emutant (Δ\u003cem\u003eAaAtg1\u003c/em\u003e/GFP-AaAtg8) strains grown in nutrient-rich conditions (PDB), nitrogen-starvation (MM-N), or 15 mM H₂O₂ for 24 h. Green fluorescence from GFP-AaAtg8 is detected to localize autophagosomes (A). Vacuoles (V) stained with CMAC dye, showing as faint blue spots. Co-localization of green and blue fluorescence indicates autophagic vacuole (AV) formation. In WT strains, autophagic vacuoles form under MM-N and H₂O₂ conditions, but not under PDB conditions. In Δ\u003cem\u003eAaAtg1 \u003c/em\u003ehyphae, autophagosomes are predominantly located outside vacuoles. Pre-autophagosomal structures (PAS, indicated by arrows) in the Δ\u003cem\u003eAaAtg1 \u003c/em\u003ehyphae grown in PDB. Scale bar = 25 μm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/748c0d3a81ce2e357b77bd3d.png"},{"id":102406966,"identity":"330ba3d4-d2c5-4551-bf35-9d5c2412b325","added_by":"auto","created_at":"2026-02-11 11:21:03","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":758140,"visible":true,"origin":"","legend":"\u003cp\u003eAaAtg1 is involved in the peroxisome degradation. Microscopic images of the wild-type (WT/mCherry-SKL) and the \u003cem\u003eAaAtg1\u003c/em\u003emutant (Δ\u003cem\u003eAaAtg1\u003c/em\u003e/mCherry-SKL) strains expressing mCherry tagged with a serine-lysine-leucine (SKL) tripeptide at the carboxyl terminus to label peroxisomes (P). Fungal hyphae were grown in PDB, MM-N, or 15 mM H₂O₂ for 24 h and examined microscopically. Distinct red fluorescent spots indicate peroxisomes, and yellow arrowheads mark vacuoles within the hyphae. Co-localization of red fluorescent spots with vacuoles indicates pexophagy. In WT, pexophagy occurs in response to MM-N and H₂O₂, but not in PDB. In contrast, Δ\u003cem\u003eAaAtg1 \u003c/em\u003efails to form pexophagy in all tested conditions. Scale bar = 25 μm.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/95957a4286e023d8f4b3f247.png"},{"id":102406608,"identity":"25553e67-8976-4725-b930-a67e86cbfb28","added_by":"auto","created_at":"2026-02-11 11:20:10","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":564663,"visible":true,"origin":"","legend":"\u003cp\u003eAaAtg1 physically interacts with AaAtg13 and AaAtg8.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) (\u003cstrong\u003eB\u003c/strong\u003e) Y2H assays. Yeast cells expressing AaAtg1-AD (prey) and AaAtg13-BD or AaAtg8-BD (Bait) successfully grew on QDO/X/A plates, indicating interaction. Positive control (T-prey × p53-bait) also supported growth, while negative controls (T-prey × Lam-bait, AaAtg1-AD × BD-empty, and AD-empty × AaAtg13-BD/AaAtg8-BD) showed no growth. (\u003cstrong\u003eC\u003c/strong\u003e) (\u003cstrong\u003eD\u003c/strong\u003e) BiFC assays. Co-expression of GFP\u003csup\u003eN\u003c/sup\u003e-AaAtg13 or GFP\u003csup\u003eN\u003c/sup\u003e-AaAtg8 with GFP\u003csup\u003eC\u003c/sup\u003e-AaAtg1 in wild-type protoplasts produced green fluorescence, confirming interactions \u003cem\u003ein vivo\u003c/em\u003e. Co-expression of GFP\u003csup\u003eN\u003c/sup\u003e and GFP\u003csup\u003eC\u003c/sup\u003e (negative control) yielded no fluorescence, while full-length GFP (positive control) displayed green fluorescence. (\u003cstrong\u003eE\u003c/strong\u003e) In Co-IP assays, strains co-expressing AaAtg1-HA and GFP-AaAtg8 were cultured in PDB for 24 h. Immunoprecipitation with GFP-Trap Agarose, followed by immunoblotting using anti-GFP and anti-HA antibodies, confirmed the interaction. Actin was used as a loading control.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/aca60d3ad9d1d20fbdda60dc.png"},{"id":102406794,"identity":"0630ddf6-6bd9-45aa-aa29-73b37db0a165","added_by":"auto","created_at":"2026-02-11 11:20:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":587359,"visible":true,"origin":"","legend":"\u003cp\u003eStructural impact of AIM deletion on AaAtg1 conformation predicted by AlphaFold2.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Schematic representation of AaAtg1 protein domains, including the kinase domain, MIT motifs, Atg13-binding region, and AIM/LIR region (EGFVFVEK). Structural models of wild-type AaAtg1 (green) and the AIM-deletion variant AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e (blue; residues 532–535 removed) were generated using AlphaFold2. Superimposition of the two models reveals a substantial overall conformational shift, with a root-mean-square deviation (RMSD) of 30.290, indicating that deletion of the AIM region causes widespread structural rearrangement.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Enlarged view of the AIM region (highlighted in red boxes). In the wild-type protein, the AIM adopts a defined orientation and side-chain positioning. In contrast, deletion of FVFV residues in AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e leads to significant distortion of the local helical structure, suggesting a loss of structural integrity required for stable Atg8 binding.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/7b95070ff618a045431120ef.png"},{"id":102406714,"identity":"7b2dff8b-41f9-4a3f-8199-8bb297f3fa98","added_by":"auto","created_at":"2026-02-11 11:20:23","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":542282,"visible":true,"origin":"","legend":"\u003cp\u003eStructural modeling of wild-type AaAtg1 and its AIM point-mutant variant. (\u003cstrong\u003eA\u003c/strong\u003e) AlphaFold2-generated structural models of wild-type AaAtg1 (green, residues 530–537: EGFVFVEK) and the AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e point-mutant (pink, EAAVFAAK), which replaces the core AIM residues G531, F532, V535, and E536 with alanine. Superimposition reveals an RMSD of 2.158, suggesting a modest global structural deviation.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eB\u003c/strong\u003e) Magnified view of the AIM region shows local conformational shifts in the AaAtg1\u003csup\u003e AAVFAA\u003c/sup\u003e mutant. Red-highlighted side chains represent the alanine substitutions. Despite overall structural preservation, mutations in AIM alter residue positioning, potentially affecting Atg8 docking and downstream autophagy regulation.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/8685493aaf6cc029da1c9cab.png"},{"id":102406899,"identity":"85079c1a-996c-43b9-9ed2-da15ef1d2af0","added_by":"auto","created_at":"2026-02-11 11:20:52","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":568665,"visible":true,"origin":"","legend":"\u003cp\u003eDeletion or mutation of the AIM region in AaAtg1 disrupts interaction with AaAtg8.\u003cstrong\u003e \u003c/strong\u003e(\u003cstrong\u003eA\u003c/strong\u003e) Y2H assays show that yeast cells expressing AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e-AD (prey) and AaAtg8-BD (bait) failed to grow on QDO/X/A plates, indicating a disrupted interaction. Positive control (T-prey × p53-bait) supports growth, while negative controls (T-prey × Lam-bait, AaAtg1ΔFVFV-AD × BD-empty, and AD-empty × AaAtg8-BD) show no growth. (\u003cstrong\u003eB\u003c/strong\u003e) Y2H assays. Yeast cells expressing AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e-AD (prey) and AaAtg8-BD (bait) fail to grow on QDO/X/A plates, confirming disrupted interaction. (\u003cstrong\u003eC\u003c/strong\u003e) BiFC assays. Co-expression of GFP\u003csup\u003eN\u003c/sup\u003e-AaAtg8 and GFP\u003csup\u003eC\u003c/sup\u003e-AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e in wild-type protoplasts results in green fluorescence, confirming the presence of a residual interaction. Co-expression of GFP\u003csup\u003eN\u003c/sup\u003e and GFP\u003csup\u003eC\u003c/sup\u003e (negative control) yields no fluorescence. (\u003cstrong\u003eD\u003c/strong\u003e) BiFC assays. Co-expression of GFP\u003csup\u003eN\u003c/sup\u003e-AaAtg8 and GFP\u003csup\u003eC\u003c/sup\u003e-AaAtg1AAVFAA in wild-type protoplasts results in green fluorescence, indicating a retained but altered interaction\u003cem\u003e in vivo\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/cf8ad3218c03ed0e5d9ba97b.png"},{"id":102406764,"identity":"c9a5ce56-db6b-47b2-bbb5-e3297d8aef5a","added_by":"auto","created_at":"2026-02-11 11:20:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":348183,"visible":true,"origin":"","legend":"\u003cp\u003eThe AIM region of AaAtg1 modulates autophagy in \u003cem\u003eA. alternata\u003c/em\u003e. (\u003cstrong\u003eA\u003c/strong\u003e) Microscopic images showing green fluorescence from the Δ\u003cem\u003eAaAtg1 \u003c/em\u003eco-expressing AaAtg1 and GFP-AaAtg8 (control strain). Δ\u003cem\u003eAaAtg1 \u003c/em\u003eco-expressing AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e and GFP-AaAtg8 resulted in two types of transformants. One group (14 transformants, exemplified by No. 14) exhibits fluorescence patterns and intensities similar to the control strain. The other group (18 transformants, exemplified by No. 18) displays weak fluorescence spots within the hyphae. Fungal strains were grown in PDB overnight and shifted to MM-N for 6 h. Scale bar = 25 μm. (\u003cstrong\u003eB\u003c/strong\u003e) Western blot analysis of GFP-AaAtg8 cleavage in the control strain, transformant No. 14, and transformant No.18. The cleavage of GFP-AaAtg8 indicates autophagic activity. (C) Western blot analysis of GFP-AaAtg8 cleavage in the control strain and Δ\u003cem\u003eAaAtg1\u003c/em\u003e transformants (No. 10 and No. 12) co-expressing AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e and GFP-AaAtg8. Fungal hyphae grown in PDB overnight were harvested, washed with sterile water, and shifted to MM-N for 6 h.\u003c/p\u003e","description":"","filename":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/3d3582a56c3a1ddd069f2331.png"},{"id":102406600,"identity":"480fa12c-8f2a-4bbf-b522-6ce8c577ff42","added_by":"auto","created_at":"2026-02-11 11:20:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":687506,"visible":true,"origin":"","legend":"\u003cp\u003eAaAtg8 mediates the recruitment and degradation of AaAtg1 in vacuoles. Microscopic images showing green fluorescence emitted by GFP-AaAtg1 in the Δ\u003cem\u003eAaAtg1\u003c/em\u003e and the Δ\u003cem\u003eAaAtg8\u003c/em\u003estrains. Conidia were grown in PDB overnight, shifted to MM-N, and incubated for 6 and 24 h. GFP-AaAtg1 is localized to vacuoles in Δ\u003cem\u003eAaAtg1\u003c/em\u003e under nitrogen starvation, while its localization is disrupted in Δ\u003cem\u003eAaAtg8\u003c/em\u003e, indicating the role of AaAtg8 in facilitating AaAtg1 degradation in vacuoles. Scale bar = 25 μm.\u003c/p\u003e","description":"","filename":"9.png","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/4e23e709e43a727cf9141612.png"},{"id":102407111,"identity":"f1d60b01-012f-4693-8fd7-d5104de61733","added_by":"auto","created_at":"2026-02-11 11:21:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6733840,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/33b063de-a52e-4bbb-a356-ec522c824b2a.pdf"},{"id":102406831,"identity":"f2ede8cf-f92b-45ac-a2f0-3399c7070c4a","added_by":"auto","created_at":"2026-02-11 11:20:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19042900,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/67162abb48a21e41b8fd57c3.docx"},{"id":102406901,"identity":"b33af657-b3e6-452b-94e6-ae108dbc1fa0","added_by":"auto","created_at":"2026-02-11 11:20:53","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":23174,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTablesS1andS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-8345089/v1/9efa713259e77e522009153f.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Autophagy-related protein 1 orchestrates autophagy initiation and feedback degradation in Alternaria alternata","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEukaryotic cells utilize autophagy, an intracellular degradation and recycling process, to maintain cellular homeostasis and adapt to environmental stress [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Autophagy is mediated by the coordinated action of an intricate machinery involving a group of autophagy-related proteins (Atgs) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The process begins at the isolation membrane, where several Atg proteins are assembled to form a pre-autophagosomal structure (PAS) (also called phagophore assembly site) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. A key player in this early step is Atg1, a serine/threonine kinase homologous to the mammalian Unc-51-like kinase 1 (ULK1) [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. In yeast, Atg1 forms a complex with Atg13 and associates with the Atg17-Atg31-Atg29 trimer to localize to the PAS and initiate autophagosome formation [\u003cspan additionalcitationids=\"CR7 CR8\" citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Subsequently, PAS expands to form double-membraned autophagosomes that engulf damaged proteins and organelles [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. This self-recycling mechanism is crucial for cell survival under stressful conditions and for maintaining cellular homeostasis under ordinary circumstances. Autophagy is required for various cellular processes in filamentous fungi, including growth, cellular differentiation, development, conidiation, appressorium formation, and virulence [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. While the importance of autophagy in nutrient limitation and stress responses is well recognized, its regulatory mechanisms, particularly the interplay among key autophagy proteins, remain incompletely understood in plant-pathogenic fungi.\u003c/p\u003e \u003cp\u003eAtg1 phosphorylation is regulated by several kinases, including the Target of Rapamycin kinase (TOR) [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], the AMP-activated protein kinase (AMPK) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and protein kinase A (PKA) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Nutrient availability critically determines TOR activity, which in turn modulates Atg1 function. Under nutrient-poor conditions, TOR is suppressed, allowing Atg13 to remain dephosphorylated and to bind Atg1 with high affinity, thereby activating autophagy [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Conversely, under nutrient-rich conditions, TOR phosphorylates Atg13, weakening its interaction with Atg1 and inhibiting autophagy. The phosphorylated Atg1 phosphorylates Atg13 to regulate autophagy initiation and directly phosphorylates Atg11 to control selective autophagy processes such as pexophagy [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This dynamic phosphorylation-dephosphorylation cycle ensures precise regulation of autophagy in response to cellular nutrient status.\u003c/p\u003e \u003cp\u003eAutophagosome membrane expansion requires the ubiquitin-like protein Atg8 [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], which conjugates to phosphatidylethanolamine (PE) and anchors to the membrane surface [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In mammals, Atg8 homologs, such as microtubule-associated protein light chain 3 (LC3) or the gamma-aminobutyric acid A receptor-associated protein (GABARAP) [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], also serve as markers for monitoring autophagy formation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Beyond facilitating membrane expansion, the Atg8-PE conjugate plays key roles in cargo selection and autophagosome-vacuole fusion [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It interacts with receptor proteins containing Atg8-family-interacting motifs (AIM) in yeast or LC3-interacting region (LIR) in mammals, thereby sequestering target proteins or damaged organelles during autophagosome expansion [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally, Atg8 recruits Atg1/ULK1 to autophagosomes, facilitating their maturation and fusion with vacuoles, a process crucial for degrading the Atg1/Atg13 complex [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This interaction relies on the AIM of Atg1, which is essential for its binding to Atg8, as demonstrated in yeast. Mutations in the AIM disrupt Atg1-Atg8 binding, impair Atg1 transport to vacuoles, and hinder autophagy [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eAlternaria alternata\u003c/em\u003e is a necrotrophic fungal pathogen that causes diseases in more than 100 plant species and obtains nutrients exclusively from dead tissues [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Many \u003cem\u003eA. alternata\u003c/em\u003e pathogens produce host-selective toxins, which are major contributors to pathogenicity [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. During host colonization, \u003cem\u003eA. alternata\u003c/em\u003e must also overcome the toxicity of reactive oxygen species (ROS) generated by the plant. We have previously shown that pexophagy, the selective autophagic degradation of peroxisomes, plays a crucial role in ROS resistance and cellular homeostasis in \u003cem\u003eA. alternata\u003c/em\u003e [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Moreover, our recent study revealed that autophagy regulates siderophore production and intracellular iron homeostasis, thereby contributing to ROS detoxification; notably, Δ\u003cem\u003eAaAtg1\u003c/em\u003e failed to produce siderophores and displayed hypersensitivity to the iron chelator bathophenanthrolinedisulfonic acid (BPS) [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, we sought further to dissect the role of AaAtg1 in stress-induced autophagy, focusing on its contributions to autophagic flux and broader regulatory functions. Our results indicate that AaAtg1 is essential for autophagy and that its degradation depends on interaction with AaAtg8. Intriguingly, mutations in the AIM of AaAtg1 disrupted its interaction with AaAtg8 but paradoxically enhanced autophagic activity in certain mutant strains. These observations point to a previously unrecognized dual role of AaAtg1 in both initiating and modulating autophagy, highlighting the complexity of autophagy regulation in filamentous fungi.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAaAtg1 is required for growth and development\u003c/h2\u003e \u003cp\u003eTo investigate the role of \u003cem\u003eAaAtg1\u003c/em\u003e in autophagy and pathogenesis in \u003cem\u003eA. alternata\u003c/em\u003e, two deletion mutant strains (designated Δ\u003cem\u003eAaAtg1\u003c/em\u003e-D6 and Δ\u003cem\u003eAaAtg1\u003c/em\u003e-D7) were generated using a split-marker approach. Compared with the wild type, Δ\u003cem\u003eAaAtg1\u003c/em\u003e exhibited slower growth, with 10% and 30% reductions in potato dextrose agar (PDA) and minimal medium (MM), respectively (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). Wild-type conidia were septated and ellipsoidal or cylindrical, whereas Δ\u003cem\u003eAaAtg1\u003c/em\u003e produced slender conidia and abundant hyphal fragments (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB, C). Germination of Δ\u003cem\u003eAaAtg1\u003c/em\u003e conidia was markedly slower than that of the wild type. Additionally, Δ\u003cem\u003eAaAtg1\u003c/em\u003e produced significantly fewer conidia and appressorium-like structures than the wild type (Supplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eD, E). Genetic complementation of Δ\u003cem\u003eAaAtg1\u003c/em\u003e with a functional \u003cem\u003eAaAtg1\u003c/em\u003e copy (Cp8 strain) restored wild-type growth, conidiation, appressorium formation, and germination.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAaAtg1 is required for autophagy\u003c/h3\u003e\n\u003cp\u003eTransmission electron microscopy (TEM) examination revealed that autophagic vacuoles (AVs) containing dense dark contents were present in the wild-type hyphae grown in MM-N (minimal medium without nitrogen) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, Δ\u003cem\u003eAaAtg1\u003c/em\u003e lacked such AVs; most of them contained no dense contents. Western blot analyses revealed that Δ\u003cem\u003eAaAtg1\u003c/em\u003e expressing GFP-AaAtg8 failed to initiate autophagy when exposed to hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) or MM-N, while the wild type accumulated free GFP, indicative of active autophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFluorescence microscopy further confirmed these findings. In wild-type hyphae incubated in MM-N or treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e for 24 h, round blue fluorescent spots [7-amino-4-chloromethylcoumarin (CMAC)-stained vacuoles] were colocalized with green fluorescent spots from GFP-AaAtg8, indicating autophagosomes within vacuoles (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In contrast, in Δ\u003cem\u003eAaAtg1\u003c/em\u003e hyphae cultured in MM-N, green fluorescent spots were observed near vacuoles but did not colocalize with them. When exposed to H₂O₂ for 24 h, Δ\u003cem\u003eAaAtg1\u003c/em\u003e hyphae exhibited blurry green fluorescence, weak CMAC staining, and abnormal hyphal morphology, suggesting cell death.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAaAtg1 is involved in peroxisome turnover\u003c/h3\u003e\n\u003cp\u003eTo determine the role of AaAtg1 in pexophagy, a mCherry tag fused with serine-lysine-leucine (SKL) tripeptides was expressed in wild-type and Δ\u003cem\u003eAaAtg1\u003c/em\u003e strains to label peroxisomes. Fluorescence microscopy revealed that in both WT/mCherry-SKL and Δ\u003cem\u003eAaAtg1\u003c/em\u003e/mCherry-SKL strains, red fluorescence (representing peroxisomes) was not localized in vacuoles when cultured in potato dextrose broth (PDB) for 24 h. In the wild-type hyphae, after being shifted to MM-N or treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, red fluorescence was observed in the vacuoles, indicating active pexophagy (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, red fluorescent spots were not localized in the vacuoles of Δ\u003cem\u003eAaAtg1\u003c/em\u003e/mCherry-SKL hyphae cultured in MM-N, suggesting impaired pexophagy. Furthermore, under H₂O₂ treatment, Δ\u003cem\u003eAaAtg1\u003c/em\u003e/mCherry-SKL hyphae exhibited deformed morphology, lacked distinct vacuoles, and showed no discernible red fluorescence, indicating severe cellular damage and disrupted peroxisomal turnover.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eAaAtg1 is involved in the detoxification of ROS\u003c/h3\u003e\n\u003cp\u003eDue to the observed shrinkage and accumulation of dense materials in Δ\u003cem\u003eAaAtg1\u003c/em\u003e hyphae exposed to H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, sensitivity tests were conducted on PDA amended with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, \u003cem\u003etert\u003c/em\u003e-butyl hydroperoxide (tBHP), or cumyl peroxide. Compared to the wild type, Δ\u003cem\u003eAaAtg1\u003c/em\u003e exhibited increased hypersensitivity to all three oxidants (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA, B). However, Δ\u003cem\u003eAaAtg1\u003c/em\u003e displayed wild-type sensitivity to single oxygen-producing compounds (rose Bengal and eosin Y) and cell wall stressors, sodium dodecyl sulfate (SDS), Congo red, and calcofluor white (data not shown). When treated with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and stained with 2'-7'-dichlorofluorescein diacetate (DCFHDA), Δ\u003cem\u003eAaAtg1\u003c/em\u003e hyphae showed stronger green fluorescence, indicating greater ROS accumulation than the wild type (Supplementary Figure \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eC). Similarly, propidium iodine (PI) staining revealed significantly stronger red fluorescence in Δ\u003cem\u003eAaAtg1\u003c/em\u003e hyphae, indicating a higher proportion of dead cells after H₂O₂ treatment.\u003c/p\u003e\n\u003ch3\u003eAaAtg1 is required for full virulence but not for host-selective toxin production\u003c/h3\u003e\n\u003cp\u003ePathogenicity tests conducted on detached calamondin leaves revealed a significant reduction of necrotic lesions after inoculation with conidial suspensions of Δ\u003cem\u003eAaAtg1\u003c/em\u003e 4 days post-inoculation (dpi) (Supplementary Figure S3A). In contrast, leaves inoculated with conidia from wild-type and CP8 strains developed necrotic lesions within the same period. Pre-wounding of leaves did not enhance lesion formation by the Δ\u003cem\u003eAaAtg1\u003c/em\u003e strain. Microscopic examination revealed that conidia from wild-type and CP8 strains germinated rapidly and formed hyphae on leaf surfaces at 1 dpi (Supplementary Figure S3B). However, conidia from Δ\u003cem\u003eAaAtg1\u003c/em\u003e showed delayed germination on leaves until 2\u0026ndash;3 dpi (data not shown), correlating with the delayed necrotic lesion development. Thin-layer chromatography (TLC) and High-performance liquid chromatography (HPLC) analyses revealed no quantitative difference in host-selective ACT toxin production between wild-type and Δ\u003cem\u003eAaAtg1\u003c/em\u003e strains (Supplementary Figure S4A, B).\u003c/p\u003e \u003cp\u003e \u003cb\u003eAaAtg1 physically interacts with both AaAtg13 and AaAtg8 in\u003c/b\u003e \u003cb\u003eA. alternata\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine whether AaAtg1 physically interacts with other core autophagy components, we performed Yeast two-hybrid (Y2H) assays using AaAtg13 and AaAtg8 as potential binding partners. Yeast cells expressing AaAtg1 fused to the GAL4 activation domain (AaAtg1-AD) and either AaAtg13 or AaAtg8 fused to the binding domain (AaAtg13-BD, AaAtg8-BD) thrived on QDO/X/A selective medium (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B), indicating direct interactions between AaAtg1 and both proteins. These interactions were further validated \u003cem\u003ein vivo\u003c/em\u003e using bimolecular fluorescence complementation (BiFC) assays. Co-expression of GFP\u003csup\u003eN\u003c/sup\u003e-AaAtg13 with GFP\u003csup\u003eC\u003c/sup\u003e-AaAtg1, or GFP\u003csup\u003eN\u003c/sup\u003e-AaAtg8 with GFP\u003csup\u003eC\u003c/sup\u003e-AaAtg1, in \u003cem\u003eA. alternata\u003c/em\u003e protoplasts led to the appearance of green fluorescent signals along the hyphal compartments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, D), while the negative control strains expressing only split GFP fragments showed no fluorescence, confirming the specificity of these interactions. To further confirm the interaction between AaAtg1 and AaAtg8 under native conditions, co-immunoprecipitation (Co-IP) was performed. Cell lysates from \u003cem\u003eA. alternata\u003c/em\u003e strains co-expressing HA-tagged AaAtg1 and GFP-tagged AaAtg8 were immunoprecipitated using anti-GFP antibodies. Western blotting detected HA-AaAtg1 in the precipitated complex, confirming its association with GFP-AaAtg8 \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Together, AaAtg1 physically interacted with both AaAtg13 and AaAtg8, supporting its central role as a scaffold protein coordinating autophagy-related protein assembly in \u003cem\u003eA. alternata\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eThe AIM region affects the AaAtg1 structure and its interaction with AaAtg8\u003c/h2\u003e \u003cp\u003eAmino acid sequence analysis revealed that AaAtg1 possesses several conserved domains associated with autophagy regulation. At the N-terminus, AaAtg1 contains a canonical serine/threonine kinase domain. In contrast, its C-terminal region harbors two microtubule-interacting and transport (MIT) domains, which are predicted to mediate interaction with AaAtg13 (Supplementary Figure S5A). Additionally, sequence inspection identified a putative AIM/LIR, characterized by the conserved sequence EGFVFVEK, located between the kinase and MIT domains (Supplementary Figure S5B). This motif could likely serve as a docking site for AaAtg8 and is conserved among Atg1 orthologs in other filamentous fungi.\u003c/p\u003e \u003cp\u003eTo evaluate the structural impact of the AIM region in AaAtg1, we used AlphaFold2 to model two variants: a deletion mutant (ΔFVFV; residues 532\u0026ndash;535 removed) and a point-mutated version (AAVFAA; E\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eGFVFVE\u003c/span\u003eK \u0026rarr; E\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eAAVFAA\u003c/span\u003eK). Superimposition with the wild-type model revealed that the ΔFVFV deletion induced a substantial global structural rearrangement (RMSD\u0026thinsp;=\u0026thinsp;30.290) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In contrast, the AAVFAA substitution caused only minor overall changes (RMSD\u0026thinsp;=\u0026thinsp;2.158) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Nonetheless, both mutants exhibited pronounced local alterations at the AIM region, notably affecting loop structure and side-chain positioning (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate whether these structural changes disrupt protein\u0026ndash;protein interactions, we performed Y2H and BiFC assays. In Y2H, neither the ΔFVFV nor AAVFAA variants showed detectable interaction with AaAtg8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, B), suggesting reduced binding affinity \u003cem\u003ein vitro\u003c/em\u003e. However, BiFC assays revealed green fluorescence in both mutant strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, D), indicating residual or altered interaction \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eThe AIM mutation could increase autophagy under certain conditions\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of the AIM in autophagy flux, Δ\u003cem\u003eAaAtg1\u003c/em\u003e co-expressing AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e (deletion) or AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e (point mutation) with GFP-AaAtg8 were further examined. The control strain, Δ\u003cem\u003eAaAtg1\u003c/em\u003e co-expressing wild-type AaAtg1 and GFP-AaAtg8, displayed uniform green fluorescence in hyphae grown in PDB and distinct fluorescent spots after being shifted to MM-N (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Δ\u003cem\u003eAaAtg1\u003c/em\u003e/AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e transformants exhibited two distinct fluorescent patterns, approximately in a 1:1 ratio. One group (14 transformants), exemplified by transformant No. 14, exhibited green fluorescent patterns and intensities similar to those of the control strain. The other group (18 transformants), represented by transformant No. 18, displayed weak fluorescent spots under MM-N. Western blot analysis revealed that free GFP cleavage from GFP-AaAtg8 was 51% in transformant No. 14, comparable to the control strain (67%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). However, transformant No. 18 exhibited nearly 90% of free GFP under MM-N conditions, indicating enhanced autophagic flux. Similarly, Δ\u003cem\u003eAaAtg1\u003c/em\u003e/AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e transformants also showed two distinct fluorescent patterns. Transformant No. 10 displayed strong fluorescence comparable to the control strain, while other transformant No. 12 exhibited weak fluorescence (data not shown). Western blot analysis confirmed\u0026thinsp;~\u0026thinsp;50% free GFP in transformant No. 10, similar to the control, but ~\u0026thinsp;97% free GFP in transformant No. 12, further supporting enhanced autophagy when the AIM region was mutated (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eLocalization of AaAtg1 to vacuoles is triggered by nitrogen starvation and mediated by AaAtg8\u003c/h3\u003e\n\u003cp\u003eTo investigate the localization of AaAtg1 during autophagy and the role of AaAtg8 in this process, GFP-AaAtg1 was expressed in Δ\u003cem\u003eAaAtg1\u003c/em\u003e and Δ\u003cem\u003eAaAtg8\u003c/em\u003e strains, and the resulting transformants were examined microscopically (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). In Δ\u003cem\u003eAaAtg1\u003c/em\u003e/GFP-AaAtg1 hyphae (complementation strain), green fluorescence was uniform after being shifted to PDB for 6 h, with no visible vacuoles. Following 6 h in MM-N, vacuoles became visible, but green fluorescence was barely detectable within them. After 24 h in PDB or MM-N, green fluorescence formed distinct patches, suggesting vacuolar localization during prolonged starvation. In contrast, Δ\u003cem\u003eAaAtg8\u003c/em\u003e/GFP-AaAtg1 hyphae emitted uniform green fluorescence with no visible vacuoles after 6 h in PDB. At 24 h, vacuoles became visible, but green fluorescence was excluded from vacuoles. Similarly, after 6 or 24 h in MM-N, vacuoles in Δ\u003cem\u003eAaAtg8\u003c/em\u003e/GFP-AaAtg1 hyphae contained no green fluorescence, indicating the requirement of AaAtg8 for the proper localization of AaAtg1 to vacuoles during autophagy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe necrotrophic pathogen \u003cem\u003eA. alternata\u003c/em\u003e produces host-selective toxins [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and cell wall-degrading enzymes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] to kill its host plant, leading to the accumulation of toxic ROS [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. \u003cem\u003eA. alternata\u003c/em\u003e must detoxify ROS to obtain nutrients from dead cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Current studies have shown the important role of AaAtg1 in ROS- and starvation-sensing, regulating autophagy initiation and cargo degradation. AaAtg1 is required for normal autophagy formation, which plays a crucial role in growth, conidiation, conidial germination, appressorial development, and pathogenesis. AaAtg1 is also responsible for autophagy-mediated peroxisome turnover. AaAtg1-mediated autophagy formation plays an important role in cellular resistance to ROS and cell survival under oxidative stress and starvation. AaAtg1 physically interacts with AaAtg8 and AaAtg13 to initiate autophagy, and AaAtg8 is required for AaAtg1 degradation in the vacuoles. We have further demonstrated that the AIM region of AaAtg1 is required for binding to AaAtg8 in Y2H but not in BiFC assays. Surprisingly, mutating the AIM region did not affect autophagy formation in some transformants but did in others. This suggests a leaky regulation in autophagy formation once the AIM region is mutated.\u003c/p\u003e \u003cp\u003eIn both yeast and mammals, the Atg1/ULK1 complex integrates nutrient starvation signals and recruits core autophagy proteins to the PAS to initiate autophagy [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, in \u003cem\u003eA. alternata\u003c/em\u003e, deletion of AaAtg1 does not abolish PAS-like structures. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, GFP-AaAtg8 puncta remain detectable under nutrient-rich conditions, appearing as small bright fluorescent dots, suggesting that AaAtg1 is not essential for the basal recruitment of Atg8 to the PAS. Under nitrogen starvation, larger GFP-AaAtg8 puncta accumulate adjacent to vacuoles in the Δ\u003cem\u003eAaAtg1\u003c/em\u003e mutant but fail to enter them, and show no GFP cleavage. These results indicate a complete block in autophagic flux once AaATg1 is impaired. This phenotype partially resembles that of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, in which Atg1 is dispensable for the initial PAS localization of Atg8 and Atg9 but is required for autophagosome formation and progression of autophagy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In \u003cem\u003eA. alternata\u003c/em\u003e, however, AaAtg1 appears to play a more stringent role. Although PAS-like puncta can form without it, subsequent steps, including autophagosome maturation, vacuolar fusion, and cargo degradation, are completely impaired. This strict dependency is corroborated by TEM and proteolytic assays, which confirm the absence of autophagic vacuoles in Δ\u003cem\u003eAaAtg1\u003c/em\u003e under both nitrogen starvation and oxidative stress.\u003c/p\u003e \u003cp\u003eΔ\u003cem\u003eAaAtg1\u003c/em\u003e is highly sensitive to H₂O₂ and other peroxides. Although PAS and autophagosomes are present in Δ\u003cem\u003eAaAtg1\u003c/em\u003e hyphae under nutrient-poor conditions (e.g., MM-N), these structures are absent following H₂O₂ treatment, likely due to ROS-induced cell death. In contrast, wild-type hyphae form autophagic vacuoles under both nitrogen starvation and oxidative stress. DCFHDA staining reveals elevated intracellular H₂O₂ accumulation in Δ\u003cem\u003eAaAtg1\u003c/em\u003e compared to the wild type, and PI staining confirms extensive cell death after H₂O₂ exposure. These findings underscore the importance of autophagy in maintaining redox homeostasis and promoting survival under oxidative stress. Previous studies show that H₂O₂ enhances peroxisome turnover in \u003cem\u003eA. alternata\u003c/em\u003e, leading to decreased peroxisome levels [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. We therefore investigated whether AaAtg1-mediated autophagy contributes to peroxisome degradation. Both nitrogen starvation and H₂O₂ induce peroxisome aggregation and translocation into vacuoles in the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In Δ\u003cem\u003eAaAtg1\u003c/em\u003e, however, peroxisomes fail to undergo these changes: under MM-N, they remain scattered outside vacuoles, and after H₂O₂ exposure, they become undetectable. Given that peroxisomes are significant sites of H₂O₂ production [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and defects in peroxisome biogenesis render fungal cells ROS-sensitive [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], the failure to degrade peroxisomes likely contributes to ROS accumulation and cell death in Δ\u003cem\u003eAaAtg1\u003c/em\u003e. Together, these results highlight the essential role of AaAtg1 in pexophagy and oxidative stress resistance.\u003c/p\u003e \u003cp\u003eIn yeast, Atg1 interacts with Atg8 in an AIM-dependent manner [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. The AIM region (EGFVFVEK) of AaAtg1 serves as a critical docking site for AaAtg8, as demonstrated by Y2H and BiFC assays. Y2H assays show that AIM mutations (ΔFVFV or EAAVFAAK) disrupt AaAtg8 binding, whereas BiFC assays reveal residual \u003cem\u003ein vivo\u003c/em\u003e interaction, suggesting that the cellular context may enable weak or transient interactions. This points to a subtle regulatory mechanism of autophagy in \u003cem\u003eA. alternata\u003c/em\u003e. Unexpectedly, AIM mutations do not uniformly suppress autophagy. Compared to wild-type, some transformants exhibit increased autophagic flux, as evidenced by stronger GFP-AaAtg8 cleavage. These findings imply that impaired AaAtg1\u0026ndash;AaAtg8 interaction may reduce AaAtg1 degradation, leading to kinase stabilization and excessive autophagy activation. This \"leaky regulation\" contrasts sharply with yeast, where AIM mutations consistently suppress autophagy [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Together, our data suggest that the AIM region in \u003cem\u003eA. alternata\u003c/em\u003e serves not only as a docking site but also as a gatekeeper that modulates autophagic intensity, highlighting a distinct regulatory mechanism that prevents detrimental hyperactivation.\u003c/p\u003e \u003cp\u003eLocalization studies reveal that AaAtg1 translocates to vacuoles under nitrogen starvation, a process that is dependent on AaAtg8. In Δ\u003cem\u003eAaAtg8\u003c/em\u003e hyphae, GFP-AaAtg1 fails to accumulate in vacuoles, indicating that AaAtg8 mediates the vacuolar sequestration and degradation of AaAtg1 during autophagy. This AaAtg8-dependent turnover likely functions as a feedback mechanism that downregulates autophagic activity and maintains homeostasis during prolonged stress. This observation aligns with findings in \u003cem\u003eS. cerevisiae\u003c/em\u003e, where Atg1 undergoes degradation via an AIM/LIR\u0026ndash;dependent mechanism [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Autophagy deficiency in Δ\u003cem\u003eAaAtg1\u003c/em\u003e correlated with marked virulence attenuation, likely due to combined effects on hyphal growth, sporulation, and ROS detoxification. In contrast, AIM mutations did not impair virulence (data not shown), yet they revealed a possible inhibitory role of the AIM-mediated interaction in restricting excessive autophagy. This suggests that the AIM region not only facilitates Atg8 binding but also serves as a checkpoint to prevent overactivation of the autophagic pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that AaAtg1 functions not only as a trigger for autophagy initiation but also as a substrate of autophagic degradation, thus constituting a negative feedback loop, a concept rarely described in plant-pathogenic fungi. The paradoxical enhancement of autophagic flux in AIM mutants may reflect the loss of this regulatory brake, a mechanism warranting further investigation. Together, these studies highlight AaAtg1 as a macromolecular scaffold that coordinates protein\u0026ndash;protein interactions, signal transduction, and autophagic flux regulation in \u003cem\u003eA. alternata\u003c/em\u003e. These insights expand our understanding of fungal stress adaptation and virulence control via autophagy, offering new perspectives on the molecular mechanisms that govern autophagy homeostasis in filamentous plant pathogens.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFungal strains, conidiation, germination, and sensitivity assays\u003c/h2\u003e \u003cp\u003eThe wild-type strain of \u003cem\u003eA. alternata\u003c/em\u003e used for transformation and mutagenesis has been previously characterized [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Fungal strains were point-inoculated onto PDA (Difco, Franklin Lakes, NJ, U.S.A.) or MM [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e] agar using a sterile toothpick and incubated at 28\u0026deg;C. For conidiation, fungal strains were grown on PDA without parafilm sealing under light for 5 days. Conidia were collected in sterile water, and germination was assessed by incubating conidia on glass slides or 96-well microtiter plates at 28\u0026deg;C for 6 h.\u003c/p\u003e \u003cp\u003eFungal sensitivity assays were conducted on PDA plates (90 mm \u0026times; 15 mm Petri dishes) containing test compounds, including calcofluor white (200 \u0026micro;M in dimethyl sulfoxide), Congo red (75 \u0026micro;M in ethanol), sodium dodecyl sulfate (SDS, 0.01%), rose Bengal (30 \u0026micro;M), eosin Y (100 \u0026micro;M), hydroperoxide (H₂O₂, 20 mM), \u003cem\u003etert\u003c/em\u003e-butyl hydroperoxide (tBHP, 3.75 mM), and cumyl hydroperoxide (3 mM in ethanol). Unless otherwise specified, all compounds were dissolved in water. Each treatment was performed in triplicate, and experiments were independently repeated three times.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic modification in\u003c/b\u003e \u003cb\u003eA. alternata\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe \u003cem\u003eAaAtg1\u003c/em\u003e sequence (accession number: KAH8628380) was retrieved from the complete genome of the \u003cem\u003eA. alternata\u003c/em\u003e EV-MIL-31 strain. Functional motifs within AaAtg1 were identified using the InterPro database and protein domain analysis tools [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A multiple sequence alignment illustrating the functional domains of the Atg1 homologs across different species was generated using the Illustrator for Biological Sequences (IBS) software [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. All Atg1 protein sequences were obtained from the National Center for Biotechnology Information (NCBI) database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.ncbi.nlm.nih.gov\u003c/span\u003e\u003cspan address=\"http://www.ncbi.nlm.nih.gov\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The AIM/LIR region was predicted using the iLIR server (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://ilir.warwick.ac.uk/\u003c/span\u003e\u003cspan address=\"https://ilir.warwick.ac.uk/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Structure models of full-length AaAtg1 and its AIM-mutated variants were constructed using AlphaFold2 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb\u003c/span\u003e\u003cspan address=\"https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold2.ipynb\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTwo Δ\u003cem\u003eAaAtg1\u003c/em\u003e mutants (D6 and D7) and a complementation strain (CP8) were previously generated and characterized [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Δ\u003cem\u003eAaAtg1\u003c/em\u003e mutants were transformed with pCB1532-GFP-AaAtg8 or pCB1532-mCherry-SKL to generate Δ\u003cem\u003eAaAtg1\u003c/em\u003e/GFP-AaAtg8 and Δ\u003cem\u003eAaAtg1\u003c/em\u003e/mCherry-SKL strains, respectively, using protoplasts from the Δ\u003cem\u003eAaAtg1\u003c/em\u003e D6 mutant. Protoplast preparation and fungal transformations were conducted as described previously [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Wild-type strains expressing GFP-AaAtg8 (WT/GFP-AaAtg8) or mCherry-SKL (WT/mCherry-SKL) from previous studies [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] were used as controls. To investigate the localization of AaAtg1 in the absence of AaAtg8, the pNEOGPE1-GFP-AaAtg1 plasmid was constructed and transformed into protoplasts of both Δ\u003cem\u003eAaAtg1\u003c/em\u003e and Δ\u003cem\u003eAaAtg8\u003c/em\u003e strains, with the latter generated in a previous study [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Oligonucleotide primers used in this study are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and all plasmids are detailed in Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eSite-directed mutagenesis and plasmid construction\u003c/h2\u003e \u003cp\u003eFungal strains were cultured in a liquid complete medium [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] on a shaker at 28\u0026deg;C for 2 days. Mycelium was harvested and ground in liquid nitrogen for RNA isolation using TRI-reagent (Sigma-Aldrich, St. Louis, MO, U.S.A.) and the PureLink RNA Kit (Invitrogen, Waltham, MA, U.S.A.) according to the manufacturer\u0026rsquo;s protocols. Complementary DNA (cDNA) was synthesized from the isolated RNA using iScript Reverse Transcriptase (Bio-Rad, Hercules, CA, U.S.A.).\u003c/p\u003e \u003cp\u003eTwo-step fusion PCR mutagenesis was performed using cDNA as a template to generate the AaAtg1 AIM deletion (AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e) and point mutation (AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e) variants. In the first step, two overlapping cDNA fragments were amplified using specific primers (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). These fragments were then used as templates for a second round of PCR to generate the full-length AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e or AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e fragment. PCR products were digested with EcoRV and KpnI and cloned into the pCB1532 vector to yield pCB1532-AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e or pCB1532-AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e. The identity of the cloned cDNA fragments was confirmed by sequencing. Plasmids were individually transformed into protoplasts prepared from the Δ\u003cem\u003eAaAtg1\u003c/em\u003e D6 mutant strain. The resulting strains were used for AaAtg8-AaAtg1 AIM binding studies. To assess the effect of AIM mutations on GFP-AaAtg8 expression and autophagy formation, a pBARS-GFP-AaAtg8 plasmid containing a bialaphos resistance gene was transformed into protoplasts prepared from the Δ\u003cem\u003eAaAtg1\u003c/em\u003e/AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e or Δ\u003cem\u003eAaAtg1\u003c/em\u003e/AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e strain.\u003c/p\u003e \u003cp\u003eFungal transformants were recovered on regeneration media supplemented with the appropriate selective agents, including bialaphos (400 \u0026micro;g/ml, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), geneticin (G418, 300 \u0026micro;g/mL, Amresco, Solon, OH, U.S.A.), or sulfonylurea (10 \u0026micro;g/ml, ChemService Inc., West Chester, PA, U.S.A.). Fungal strains were further screened and validated by PCR using specific primers (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eWestern blot analysis\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed as previously described [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Briefly, fungal strains were cultured in potato dextrose broth (PDB, Difco) for 24 h, then transferred to PDB, MM-N (MM without nitrogen), or 15 mM H₂O₂, and incubated for 4 h. Total proteins were extracted from mycelium using RIPA cell lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and 1% protease inhibitor cocktail (Biokit, Toufen City, Miaoli, Taiwan). Proteins were boiled in a loading buffer, separated by SDS-PAGE, and transferred onto PVDF membranes. Membranes were stained with Ponceau S to verify the transfer, blocked with Towbin\u0026rsquo;s (TSW) buffer containing 25 mM Tris, 192 mM Glycine, and often 20% methanol and 0.1% SDS, and incubated overnight at 4\u0026deg;C with a rabbit anti-GFP antibody (1:5000). After washing, membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000, Jackson ImmunoResearch, West Grove, PA, U.S.A.). Signals were detected using LumiFlash Prime Chemiluminescent Substrate, HRP (Visual Protein, Taipei, Taiwan). Chemiluminescence images were captured with the ChemiDoc MP imaging system (Bio-Rad) and analyzed using ImageLab software (version 6.1.0, Bio-Rad). Band intensity was quantified using ImageJ software (US National Institutes of Health, Bethesda, MD, U.S.A.) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://imagej.net/ij/\u003c/span\u003e\u003cspan address=\"https://imagej.net/ij/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCo-immunoprecipitation (Co-IP)\u003c/h2\u003e \u003cp\u003eFor Co-IP experiments, an \u003cem\u003eAaAtg1\u003c/em\u003e fragment was amplified using primers ATG1 F EcoRV and ATG1 R KpnI, digested with EcoRV and KpnI, and cloned into the pNEOGPE1-HA plasmid containing a linker HA-tag sequence (5\u0026rsquo;-atgggcagctacccatacgatgttccagattacgct-3\u0026rsquo;) to generate pNEOGPE1-HA-AaAtg1. The plasmids pNEOGPE1-HA-AaAtg1 and pCB1532-GFP-AaAtg8 were individually or co-transformed into wild-type protoplasts, resulting in three distinct fungal strains. Fungal strains were cultured in PDB for 24 h, and total proteins were extracted using RIPA buffer amended with Ser/Thr phosphatase inhibitors (1.0 mM Na\u003csub\u003e3\u003c/sub\u003eVO\u003csub\u003e4\u003c/sub\u003e and 1.0 mM/l NaF; Sigma-Aldrich). Immunoprecipitation was performed using GFP-Trap Agarose beads (Chromotek, Islandia, NY, U.S.A.) according to the manufacturer\u0026rsquo;s instructions. Proteins were eluted from magnetic beads using 2\u0026times; Laemmli buffer, and 25 \u0026micro;l of the sample was subjected to western blot analysis. Actin served as the internal control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid (Y2H) assays\u003c/h2\u003e \u003cp\u003eY2H assays were conducted using the GAL4-based Matchmaker Yeast Two-Hybrid System (Clontech Laboratories, Mountain View, CA, U.S.A.). Full-length coding regions of AaAtg1, AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e, and AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e were individually amplified, fused with GAL4 activation domain (AD), and cloned into the pGADT7 plasmid to generate preys (AaAtg1-AD, AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e-AD, and AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e-AD). Similarly, the full-length coding regions of AaAtg8 and AaAtg13 were fused to the GAL4 DNA-binding domain (DBD) and cloned into the pGBKT7 vector as baits (AaAtg8-BD and AaAtg13-BD).\u003c/p\u003e \u003cp\u003ePlasmids were propagated in \u003cem\u003eEscherichia coli\u003c/em\u003e and introduced into the yeast Y187 or Y2HGold strains according to the manufacturer\u0026rsquo;s protocol (Clontech). Negative (T-prey x Lam-bait) and positive (T-prey x p53-bait) controls were included to validate the assay. Four independent transformants for each experimental pairing were tested. Yeast strains were plated on selective SD/\u0026ndash;Leu/\u0026ndash;Trp/\u0026ndash;Ade/\u0026ndash;His agar plates containing X-α-Gal (40 \u0026micro;g/ml) and Aureobasidin A (200 ng/ml) (QDO/X/A). Growth on the selective medium indicated a strong interaction between the tested proteins. Primers used for plasmid construction are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, with restriction enzyme recognition sites incorporated to facilitate cloning.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eBimolecular fluorescence complementation (BiFC) assay\u003c/h2\u003e \u003cp\u003eThe N-terminal half of GFP (GFP\u003csup\u003eN\u003c/sup\u003e) was amplified from pCB1532-GFP-AaAtg8 by PCR and subcloned into pHygroGPE1 to yield pHygroGPE1-GFP\u003csup\u003eN\u003c/sup\u003e. The C-terminal half of GFP (GFP\u003csup\u003ec\u003c/sup\u003e) was cloned into pNEOGPE1 to result in pNEOGPE1-GFP\u003csup\u003eC\u003c/sup\u003e. Full-length coding sequences of AaAtg1, AaAtg1\u003csup\u003eΔFVFV\u003c/sup\u003e, and AaAtg1\u003csup\u003eAAVFAA\u003c/sup\u003e were individually inserted into the pNEOGPE1-GFP\u003csup\u003eC\u003c/sup\u003e vector to generate three plasmid constructs (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Similarly, AaAtg8 and AaAtg13 coding sequences were individually cloned into pHygroGPE1-GFP\u003csup\u003eN\u003c/sup\u003e. The resulting plasmid pairs were co-transformed into wild-type protoplasts, and transformants were recovered on regeneration medium plates supplemented with 200 \u0026micro;g/ml hygromycin and 300 \u0026micro;g/ml geneticin. Transformants were confirmed by PCR. After incubation on PDA for 3 days, hyphae were examined microscopically for green fluorescence to evaluate GFP reconstitution.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eToxin production and fungal virulence\u003c/h2\u003e \u003cp\u003eFungal strains were cultured in modified Richard's medium [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] for 21 days, and culture filtrates were collected for ACT purification using Amberlite XAD-2 resin (Sigma-Aldrich). ACT was dissolved in methanol and analyzed by TLC and HPLC, as described previously [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Fungal pathogenicity was evaluated by applying 10 \u0026micro;l of conidial suspensions (1\u0026times;10⁵ cells/ml) onto detached calamondin (\u003cem\u003eCitrofortunella mitis\u003c/em\u003e) leaves, either unwounded or wounded with a fine needle. Leaves treated with sterile water served as a mock control. Treated leaves were incubated in a plastic box for 3\u0026ndash;4 days to observe necrotic lesion development. Each fungal strain was tested on at least four leaves, and the experiments were repeated 3 times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eMicroscopy\u003c/h2\u003e \u003cp\u003eHyphae were stained with lactophenol-cotton blue (Sigma-Aldrich) in ethanol (4:1, v/v) for 20 min and observed using a Nikon Optiphot 2 Microscope (Tokyo, Japan). CMAC (7-amino-4-chloromethylcoumarin, 100 \u0026micro;M; Thermo Fisher Scientific, Waltham, MA, U.S.A.) was used to stain vacuoles. DCFHDA (2'-7'-dichlorofluorescein diacetate, Sigma-Aldrich) was used to detect cellular ROS. Cell viability was assessed using propidium iodide (PI, Sigma-Aldrich) staining. Fluorescence was detected using a ZOE Fluorescent Cell Imager (Bio-Rad) with appropriate excitation and emission wavelengths, as previously described [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. TEM was performed as described [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. All experiments were repeated at least twice.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using SPSS Statistics 20 software (IBM, NY, U.S.A.). Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The homogeneity of variance was assessed with Levene\u0026rsquo;s test. Statistical significance was evaluated using either an independent sample \u003cem\u003et\u003c/em\u003e-test or one-way ANOVA, followed by Tukey\u0026rsquo;s honest significant difference (HSD) post hoc test (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Each treatment contained at least three replicates unless otherwise specified.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAaAtg \u003cem\u003eAlternaria alternata\u003c/em\u003e autophagy-related protein\u003c/p\u003e\n\u003cp\u003eAIM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Atg8-family-interacting motif\u003c/p\u003e\n\u003cp\u003eROS\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Reactive oxygen species\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePDB\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Potato dextrose broth\u003c/p\u003e\n\u003cp\u003eMM-N\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Minimal medium without nitrogen\u003c/p\u003e\n\u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Hydrogen peroxide \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eACT \u003cem\u003eAlternaria citri\u003c/em\u003e tangerine toxin\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe original contributions presented in the study are included in the article/Supplementary Material; further inquiries can be directed to the corresponding authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have declared that no competing interests exist.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Science and Technology Council, Taiwan [grant numbers 112-2313-B-005-033 to K.-R. Chung, and 110-2326-B-005-001-MY3 to P.-C. Wu and K.-R. Chung]; intramural funding from China Medical University (project number CMU113-N-09 to P.-C. Wu); and the Ministry of Education, Taiwan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCYLC and HYL performed the experiments, analyzed the data, validated the results, and reviewed and edited the manuscript. KRC and PCW designed the experiments, sought funding, provided supervision, validated the results, and wrote the original draft. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLahiri V, Hawkins WD, Klionsky DJ. Watch what you (self-) eat: autophagic mechanisms that modulate metabolism. 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Alternaria host-specific (HSTs) toxins: An overview of chemical characterization, target sites, regulation and their toxic effects. Toxicol Rep. 2019;6:745-58. doi: 10.1016/j.toxrep.2019.06.021.\u003c/li\u003e\n \u003cli\u003eWu P-C, Chen Y-K, Yago JI, Chung K-R. Peroxisomes implicated in the biosynthesis of siderophores and biotin, cell wall integrity, autophagy, and response to hydrogen peroxide in the citrus pathogenic fungus \u003cem\u003eAlternaria alternata\u003c/em\u003e. Front Microbiol. 2021;12:645792. doi: 10.1111/mpp.13247.\u003c/li\u003e\n \u003cli\u003eWu P-C, Choo CYL, Lu H-Y, Wei X-Y, Chen Y-K, Yago JI, et al. Pexophagy is critical for fungal development, stress response, and virulence in \u003cem\u003eAlternaria alternata\u003c/em\u003e. Mol Plant Pathol. 2022;23(10):1538-54. doi: 10.3389/fmicb.2021.645792.\u003c/li\u003e\n \u003cli\u003eWu P-C, Choo Y-L, Wei S-Y, Yago JI, Chung K-R. Contribution of Autophagy to Cellular Iron Homeostasis and Stress Adaptation in \u003cem\u003eAlternaria alternata\u003c/em\u003e. Int J Mol Sci. 2024;25(2):1123. doi: 10.3390/ijms25021123.\u003c/li\u003e\n \u003cli\u003eKohmoto K, Itoh Y, Shimomura N, Kondoh Y, Otani H, Kodama M, et al. Isolation and biological activities of two host-specific toxins from the tangerine pathotype of \u003cem\u003eAlternaria alternata\u003c/em\u003e. Phytopathology. 1993;83(5):495-502. doi: 10.1094/PHYTO-83-495.\u003c/li\u003e\n \u003cli\u003eMa H, Zhang B, Gai Y, Sun X, Chung K-R, Li H. Cell-wall-degrading enzymes required for virulence in the host selective toxin-producing necrotroph \u003cem\u003eAlternaria alternata\u003c/em\u003e of citrus. Front Microbiol. 2019;10:2514. doi: 10.3389/fmicb.2019.02514.\u003c/li\u003e\n \u003cli\u003eLin C-H, Yang SL, Chung K-R. 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J Fungi. 2020;6(4):248. doi: 10.3390/jof6040248\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"AIM, Peroxisome turnover, Nutrient sensing, Oxidative stress tolerance, Fungal pathogenicity, Autophagic flux regulation","lastPublishedDoi":"10.21203/rs.3.rs-8345089/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8345089/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAutophagy plays an essential role in fungal development and stress adaptation, yet its regulatory mechanisms in filamentous fungi remain incompletely understood. We functionally characterized \u003cem\u003eAlternaria alternata\u003c/em\u003e Atg1 (AaAtg1), a serine/threonine kinase, and demonstrated its dual roles in autophagy initiation and flux modulation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDeletion of \u003cem\u003eAaAtg1\u003c/em\u003e abolishes autophagosome formation and autophagic flux, impairs peroxisome degradation, and leads to hypersensitivity to oxidative stress, as well as reduced virulence. AaAtg1 physically interacts with core autophagy proteins AaAtg13 and AaAtg8, and its vacuolar degradation is AaAtg8-dependent. Structure-guided mutagenesis of the Atg8-family interacting motif (AIM) disrupts AaAtg1\u0026ndash;AaAtg8 binding in yeast two-hybrid assays but not in bimolecular fluorescence complementation, suggesting partial functional retention \u003cem\u003ein vivo\u003c/em\u003e. Intriguingly, AIM mutations do not impair autophagy; instead, some transformants exhibit elevated autophagic activity, suggesting a potential negative regulatory role of AIM in autophagy tuning.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese findings reveal a noncanonical feedback mechanism in which AaAtg8 facilitates AaAtg1 degradation to modulate autophagic output. Our study elucidates the structure\u0026ndash;function relationship of AaAtg1 and uncovers a dual regulatory mechanism that coordinates autophagy progression and stress adaptation in the plant-pathogenic fungus.\u003c/p\u003e","manuscriptTitle":"Autophagy-related protein 1 orchestrates autophagy initiation and feedback degradation in Alternaria alternata","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 11:14:36","doi":"10.21203/rs.3.rs-8345089/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-19T19:43:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-18T20:16:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-13T00:24:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"5834074948830727473011166409324878308","date":"2026-02-11T12:17:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"85130624219796713593842589507003291116","date":"2026-02-08T02:16:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326128744317504696538209405439345121640","date":"2026-02-06T16:21:27+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-06T10:51:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-25T20:33:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-17T03:37:18+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-16T16:59:55+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Microbiology","date":"2025-12-15T23:02:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mcro","sideBox":"Learn more about [BMC Microbiology](http://bmcmicrobiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/mcro","title":"BMC Microbiology","twitterHandle":"#bmcmicrobiology","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b79b41c2-e7ce-44a5-9021-1867a01d4a73","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-15T05:09:30+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 11:14:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8345089","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8345089","identity":"rs-8345089","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}