Nuclear basket nucleoporin MoNup50 is essential for fungal development, pathogenicity, and autophagy in Magnaporthe oryzae

Nuclear basket nucleoporin MoNup50 is essential for fungal development, pathogenicity, and autophagy in Magnaporthe oryzae | 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 Nuclear basket nucleoporin MoNup50 is essential for fungal development, pathogenicity, and autophagy in Magnaporthe oryzae Ying-Ying Cai, Xue-Ming Zhu, Muhammad Noman, Jing Wang, Zhong-Na Hao, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5584909/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 May, 2025 Read the published version in Cell Communication and Signaling → Version 1 posted 8 You are reading this latest preprint version Abstract Autophagy is crucial for appressorium development and host invasion by phytopathogenic fungi, including Magnaporthe oryzae . During appressorium maturation, many organelles, such as nuclei, in the conidia need to be degraded through autophagy to be recycled in the appressorium. However, the interplay between autophagy and nuclear membrane systems remains poorly understood. In this study, we functionally characterized MoNup50, a nuclear pore-associated protein. Despite sharing limited sequence identity with human and yeast Nup proteins, MoNup50 contains conserved domains typical of nuclear pore complex proteins. Observation under fluorescence microscopy revealed that MoNup50 localizes to the nuclear membrane in M. oryzae . Deletion of MoNUP50 resulted in reduced hyphal growth, spore production, appressorium formation, and pathogenicity, while increasing sensitivity to osmotic stress and cell wall disruption. Notably, MoNup50 interacts with the key autophagy protein MoAtg7, which regulates MoAtg8-PE synthesis during autophagy. Moreover, MoNUP50 deletion led to elevated autophagy levels and increased phosphorylation of the MAPKs Osm1 and Mps1. These findings suggest that MoNup50 is involved in appressorium morphogenesis and pathogenicity by modulating autophagy and MAPK pathways, highlighting the critical role of nuclear pore proteins in M. oryzae pathogenicity and their potential cross-talk with autophagic and MAPK signaling. rice blast fungus virulence Atg7 nuclear pore complex Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Introduction Rice blast, caused by the fungal pathogen Magnaporthe oryzae , is one of the most devastating rice diseases, often referred to as the “cancer of rice”. It seriously affects global food production and security [ 1 , 2 ] . M. oryzae primarily infects rice through conidia, which are dispersed by wind and rain. Upon landing on the host leaf surface, the conidium germinates to form an infection structure called an appressorium [ 3 ] . During appressorium maturation, glycogen and lipid droplets in the conidia translocate to the appressoria. Subsequently, these glycogen and lipid droplets are gradually degraded in the appressoria, leading to the accumulation of substantial glycerol and turgor pressure as high as 8.0 MPa [ 4 ] . This turgor pressure drives the formation of penetration pegs, enabling the fungus to breach the host leaf surface and establish infection [ 5 ] . Autophagy is a conserved cellular process in which autophagosomes engulf damaged macromolecules and organelles, transporting them to vacuoles or lysosomes for degradation [ 6 , 7 ] . This process maintains cellular homeostasis by recycling macromolecules to replenish energy and material [ 8 , 9 ] . During appressorium maturation, conidia undergo autophagy-dependent nuclear degradation, leading to conidial cell death and recycling of conidial contents into appressorium to facilitate turgor pressure generation [ 10 ] . Autophagy is regulated by a series of autophagy-related genes ( ATG s), including ATG7 , which regulates autophagy by facilitating the formation of autophagosomes through the Atg8-PE synthesis pathway [ 11 ] . Nuclear pore complexes (NPCs) are large protein assemblies embedded in the nuclear envelope, mediating the transport of biomolecules between the cytoplasm and nucleus [ 12 , 13 ] . NPCs consist of seven substructures formed by approximately 30 different nucleoporins (Nups), playing a particularly dynamic structural and functional role. In Saccharomyces cerevisiae , Nup2/Nup50 regulates nuclear transport and interacts with chromatin to influence gene expression [ 12 , 14 ] . In filamentous fungi, Nup2/Nup50 is critical for viability and stress response [ 15 ] . Nup2/Nup50 also interacts with active genes in the nucleoplasm, participates in gene regulation, and controls chromatin epigenetic states by preventing the spread of repressive marks [ 16 ] . For example, in Aspergillus nidulans , Nup2/Nup50 is critical for the cell viability [ 17 ] . Similarly, Nup2/Nup50 has been reported to be involved in growth and development, stress response, pathogenicity, and DON toxin synthesis in Fusarium graminearum [ 18 ] . Recent studies linked the NPC complex to autophagy regulation. In S. cerevisiae , TORC1 (Tor kinase complex 1) inhibition triggers autophagic degradation of NPC complex and Nups, mediated by selective autophagic receptors such as Atg8 [ 19 ] . In mammals, the nuclear basket Nup, Tpr, regulates autophagy by modulating the export of mRNAs and interacting with autophagy factors [ 20 ] . Despite extensive studies in yeast and other organisms, the role of Nup50/Nup2 in M. oryzae , especially in autophagy, remains underexplored. In this study, we characterized MoNup50, a nuclear pore-associated protein, essential for hyphal growth, sporulation, appressorium formation, and pathogenicity. MoNup50 also responds to osmotic and cell wall stress by regulating the Osm1 and Mps1 MAPK signaling pathways. Notably, MoNup50 interacts with the key autophagy protein MoAtg7 to regulate autophagy, and deletion of MoNUP50 resulted in abnormal autophagic activity. Results Identification and structural analysis of MoNup50 Structural domain analysis revealed that MoNup50 contains the conserved nuclear pore complex protein domain, the FG-repeat region, and the Ran-binding domain (Figure. S1A). This suggests a high degree of conservation of Nup50 across species, including M. oryzae . Structural predictions of the Nup50 protein indicated similar α-helix structures and disordered FG sequences across different organisms, including yeast, pathogenic fungi, and mammals (Figure. S1B). Phylogenetic analysis of Nup50 from various organisms showed that MoNup50 of M. oryzae is more closely related to that of F. graminearum , than to S. cerevisiae , Schizosaccharomyces pombe , and mammals (Figure. S1C). MoNup50 localizes to the nuclear envelope in M. oryzae To determine the subcellular localization of MoNup50 in M. oryzae , we constructed a MoNup50-GFP fusion vector and transformed it into Δ Monup50 (Fig. S1 ). The observation under fluorescence microscopy showed that MoNup50-GFP localized around the nucleus. Co-localization of MoNup50-GFP at different developmental stages (hyphae, conidia, and appressoria) with the nuclear marker protein MoH 2 B-mCherry showed that MoNup50-GFP formed a punctate structure around the nucleus in hyphae and a ring-like structure in conidia and mature appressoria, suggesting MoNup50 localizes at the nuclear envelope (Figs. 1 A and B). MoNup50 regulates hyphal growth, spore production, and spore morphology To assess the biological function of MoNup50, we created a MoNUP50 deletion strain (Δ Monup50 ) and a complemented strain (Δ Monup50 :: MoNUP50 ) (Figure S2). The Δ Monup50 mutant showed a 41% reduction in colony growth diameter compared to the wild-type (Guy11) and complemented strains (Fig. 2 A and 2 D). Sporulation assays revealed that the Δ Monup50 mutant failed to produce conidia after 24 h, with sporulation reduced by 98% compared to the Guy11 and complemented strains (Fig. 2 B and 2 E). Additionally, the conidia from the Δ Monup50 mutant displayed abnormal morphology, with over 85% showing either no septa or a single septum, whereas more than 90% of Guy11 conidia exhibited two septa (Fig. 2 C and 2 F). These results indicate that MoNup50 is essential for vegetative growth, spore morphology, and sporulation. MoNup50 regulates appressorium formation and appressorium turgor pressure At 4, 8, and 12 hours post-inoculation (hpi), the spores of Δ Monup50 mutant exhibited significantly lower appressorium formation rates compared to the Guy11 and complemented strains (Fig. 3 A and 3 C). 9.70% of the conidia in the Δ Monup50 mutant formed appressoria at 4 hpi, compared to over 50% in the Guy11 and complemented strains. At 24 hpi, however, appressorium formation rates were similar between the Δ Monup50 mutant and the Guy11. When submerged in a highly concentrated glycerol solution, the collapse rate of appressoria in the Δ Monup50 mutant was significantly higher than in the Guy11 and complemented strains, indicating the mutant’s appressoria has lower turgor pressure (Fig. 3 B and 3 D). These findings suggest that MoNup50 is crucial for appressorium formation and turgor pressure generation. MoNup50 is involved in infection and pathogenicity To examine the role of MoNup50 in pathogenicity, we first inoculated the Δ Monup50 , Guy11, and complemented strains onto detached barley leaves. At 4 days post-inoculation (dpi), the Δ Monup50 mutant showed few tiny infection lesions, while the Guy11 and complemented strains caused typical coalescent lesions (Fig. 4 A). Similarly, spore suspensions were inoculated onto barley leaves producing only small and restricted lesions by the Δ Monup50 mutant, while the Guy11 and complemented strains caused large and coalescent lesions (Fig. 4 B). In rice seedling infection assays, the Δ Monup50 mutant caused significantly fewer lesions (lesion area 11.11 ± 0.73%) compared to the Guy11 (78.98 ± 3.26%) and complemented strains (77.32 ± 2.22%) (Fig. 4 C and 4 E). Further analysis of barley leaf infection experiments at 36 and 48 hpi revealed that Δ Monup50 has defects in plant penetration and invasive growth. The infection hyphae (IH) were classified into three types, Type I (no IH), and Type II (IH in one cell), Type III (IH penetrating adjacent cell). At 48 hpi, the Δ Monup50 mutant predominantly formed Type I and Type II infection hyphae with significantly less production of Type III hyphae (~ 16%), whereas the Guy11 and complemented strains formed Type III hyphae (~ 90%) capable of spreading to adjacent cells (Fig. 4 D and 4 F). These results indicate that MoNup50 is crucial for full infection and pathogenicity. MoNup50 affects stress tolerance and signal transduction pathways To investigate the role of MoNup50 in environmental stresses, we conducted sensitivity assays in response to osmotic stressors (sorbitol, NaCl, and KCl) and cell wall stressors (CFW, SDS, and CR). We found that the Δ Monup50 mutant exhibited increased sensitivity to ionic hyperosmotic stress (NaCl and KCl) and cell wall stress (CFW, SDS, and CR), but no significant change in response to non-ionic hyperosmotic stress (Sorbitol) (Fig. 5 A, 5 B, S6A and S6C). The Osm1 MAPK and Mps1 MAPK signaling pathways are central pathways to hyperosmotic and cell wall stress responses in M. oryzae [ 21 ] . We examined the phosphorylation levels of Osm1 and Mps1 in the Guy11 and Δ Monup50 mutant. Phosphorylation levels of Osm1 and Mps1 were higher in the Δ Monup50 mutant than in the Guy11 under normal and NaCl- or CR-induced conditions, indicating that MoNup50 modulates these signaling pathways during the stress response (Fig. 5 C and 5 D, 6 B and 6 D). These findings suggest that MoNup50 responds to hyperosmotic stress and cell wall stress by regulating the Osm1 and Mps1 MAPK signaling pathways. MoNup50 affects the transport and degradation of glycogen and lipid droplet In M. oryzae , glycogen and lipid droplets in conidia are degraded and transported to the appressorium via the germ tube, leading to the accumulation of glycerol, which generates the necessary turgor pressure for infection [ 22 ] . To assess the cause of reduced turgor pressure in the Δ Monup50 mutant, we examined the transport and degradation of glycogen and lipid droplets during appressorium development in three strains. At the conidial stage, no significant difference in glycogen synthesis was observed between the Δ Monup50 mutant and the Guy11. At 8 hpi, 54.12 ± 5.44% conidia of the Δ Monup50 mutant contained glycogen, whereas only 15.51 ± 3.38% conidia of the Guy11 contained glycogen. At 16 hpi, 93.67% ± 0.65% of the glycogen in the Guy11 appressoria had been degraded, in contrast, less than 30% of the glycogen was degraded in the Δ Monup50 mutant. These data indicate that MoNup50 regulates glycogen transfer and degradation in the conidia and appressoria of M. oryzae (Fig. 7 A, 7 B, and 7 C). Intriguingly, there was no significant difference in the transport of lipid droplets in the conidia between the Δ Monup50 mutant and Guy11. However, lipid droplet degradation in the Δ Monup50 mutant appressoria was delayed (Fig. 7 D, 7 E, and 7 F). These findings suggest that MoNup50 is crucial for the transport of glycogen and the degradation of glycogen and lipid droplets in the appressorium. MoNup50 negatively regulates autophagy Tor is a key regulator of the autophagy process [ 23 ] . Rapamycin is a specific inhibitor of Tor kinase [ 24 ] . To investigate the role of MoNup50 in autophagy, we inoculated mycelial plugs of Guy11 and Δ Monup50 mutant onto plates containing 100 ng/mL rapamycin and assessed their growth. The Δ Monup50 mutant showed stronger sensitivity than Guy11 and Δ Monup50 :: MoNUP50 (Fig. 8 A and 8 B). These results suggest that MoNup50 plays a role in regulating autophagy. To further investigate whether MoNup50 directly influences autophagy, we introduced an autophagy marker GFP-MoAtg8 into the Guy11 and the Δ Monup50 mutant. We examined the subcellular localization of GFP-MoAtg8 and free GFP (which results from the degradation of GFP-MoAtg8 within vacuoles) using a fluorescence microscope (Fig. 8 C). Under nutrient-sufficient conditions, the GFP-MoAtg8 was mainly localized in the cytoplasm in the Guy11 hyphae, appearing as bright puncta near the vacuoles. In contrast, in the Δ Monup50 mutant hyphae, most of the GFP-MoAtg8 entered the vacuoles, with only a few puncta remaining near the vacuoles in the cytoplasm. At 1 h post-starvation, the Guy11 hyphae exhibited GFP-MoAtg8 puncta near the vacuoles, with additional degradation into free GFP within the vacuoles. However, in the Δ Monup50 mutant hyphae, all GFP-MoAtg8 degraded into free GFP diffusely distributed within the vacuoles, with no punctate fluorescence observed in the cytoplasm. These observations suggest that the autophagy level is higher in the Δ Monup50 mutant than in the Guy11. Autophagy is also crucial for conidia germination, during which intracellular proteins, macromolecules, and damaged organelles are degraded for cellular recycling. We assessed the autophagy level in the Δ Monup50 mutant at spore germination stages by monitoring GFP-MoAtg8 localization (Fig. 8 D). Under nutrient-sufficient conditions, GFP-MoAtg8 in the Guy11 was mainly distributed as punctate fluorescence in the cytoplasm, with minimal diffuse fluorescence of free GFP in the vacuoles, indicating a low autophagy level. In contrast, in the Δ Monup50 mutant, only a few punctate GFP-MoAtg8 signals were observed in the cytoplasm, with the majority degrading into free GFP in the vacuoles, indicating a higher autophagy level. At 1–2 h post-starvation, the Guy11 exhibited a decrease in punctate GFP-MoAtg8 signals and an increase in free GFP signals within vacuoles, indicating an increase in autophagy with prolonged starvation. In contrast, the Δ Monup50 mutant displayed almost no punctate GFP-MoAtg8 in the cytoplasm, with the majority of GFP-MoAtg8 degrading into free GFP and accumulating in vacuoles, reflecting a sustained high level of autophagy. To quantitatively analyze autophagic flux, we performed western blotting to detect GFP-MoAtg8 and free GFP protein levels (Fig. 8 E). Under nutrient-rich conditions, a strong band for GFP-MoAtg8 was observed in the Guy11, and free GFP was barely detectable. In contrast, GFP-MoAtg8 and free GFP bands were detected at comparable intensities in the Δ Monup50 mutant. Specifically, 3% of GFP-MoAtg8 in the Guy11 degraded to free GFP, while 40% of GFP-MoAtg8 degraded to free GFP in the Δ Monup50 mutant. At 1 h post-starvation, the band for free GFP in the Guy11 remained weaker than the GFP-MoAtg8 band, but at 2 h post-starvation, the free GFP band became comparable to the GFP-MoAtg8 band. In contrast, in the Δ Monup50 mutant, the free GFP band was stronger than the GFP-MoAtg8 at 1 and 2 h post-starvation. Precisely, 43% and 63% of GFP-MoAtg8 degraded into free GFP in the Guy11 at 1 and 2 h post-starvation, respectively. While 74% and 77% of GFP-MoAtg8 degraded into free GFP in the Δ Monup50 mutant, respectively. These results indicate that autophagy levels were significantly higher in the Δ Monup50 mutant than in the Guy11 under nutrient-rich and starvation conditions. MoNup50 interacts with the key autophagy protein MoAtg7 in vitro and in vivo To further validate the relationship between MoNup50 and autophagy, we screened the interaction between MoNup50 and key autophagy protein, MoAtg7. In yeast two-hybrid assays, we found that co-transforming yeast cells with the MoAtg7-AD plasmid and MoNup50-BD plasmid allowed yeast growth on SD-Leu-Trp-His-Ade plates. However, when the MoAtg7-AD plasmid was co-transformed with the pGBKT7 plasmid or the MoAtg7-AD plasmid was co-transformed with the pGADT7 plasmid into the yeast cells, no growth was observed on SD-Leu-Trp-His-Ade plates, indicating that MoNup50 interacts specifically with MoAtg7 without any self-activation (Fig. 9A). In vitro GST-pull down assays further confirmed the interaction between MoNup50 and MoAtg7. GST-MoAtg7 could pull down His-MoNup50 from the lysate using GST beads, whereas the negative control (empty GST protein) did not pull down any protein (Fig. 9B). These results indicate that MoNup50 and MoAtg7 interact directly in vitro . Additionally, we used the bimolecular fluorescence complementation (BiFC) method to confirm the interaction between MoNup50 and MoAtg7 in vivo . The C-terminal fragment of MoNUP50 was fused to the pKD5-YFPC vector, and the N-terminal fragment of MoATG7 was fused to the pKD5-YFPN vector. Both constructs were co-transformed into Guy11. As negative controls, we co-transformed YFPN/MoNup50-YFPC and YFPC/YFPN-MoAtg7 into Guy11. YFP signals were detected in the transformants expressing MoNup50-YFPC/YFPN-MoAtg7, but no fluorescence was observed in the negative controls, confirming that MoNup50 interacts with MoAtg7 in vivo (Fig. 9C). These results demonstrate that in M. oryzae , MoNup50 interacts with MoAtg7 in vitro and in vivo . MoNup50 promotes the MoAtg8-PE synthesis To investigate how MoNup50 affects MoAtg7 and its role in the autophagy process, we assessed the relative expression level of MoAtg7 in the Guy11 and the Δ Monup50 mutant. We found that the expression level of MoAtg7 in the Δ Monup50 mutant was ~ 2-fold higher than that in the Guy11 (Fig. 10 A), suggesting that Monup50 is involved in the regulation of MoAtg7 expression. In the autophagy induction process of S. cerevisiae , Atg8 undergoes lipidation, a process catalyzed by a series of enzymes, including Atg4, Atg7, Atg3, and the Atg5-Atg12-Atg16 complex. This results in the formation of Atg8-PE, which is anchored to the autophagosome membrane [ 25 , 26 ] . Given the close correlation between MoAtg8-PE and the number of autophagosomes [ 27 ] , we measured the turnover of endogenous MoAtg8 and MoAtg8-PE in the Guy11 and the Δ Monup50 mutant by western blotting to assess autophagy levels. Results showed that MoAtg8 lipidation levels increased over time in the Guy11 and the Δ Monup50 mutant under starvation conditions (Fig. 10 B). Notably, under nutrient-rich and starvation conditions, the MoAtg8-PE bands were more prominent in the Δ Monup50 mutant compared to the Guy11. At 2 h post-starvation, the MoAtg8-PE/GAPDH ratio in the Δ Monup50 mutant reached 1.55, whereas in the Guy11, the ratio was only 0.56 (Fig. 10 B). These results suggest that MoNup50 negatively regulates the conversion process of MoAtg8 to MoAtg8-PE. Discussion Eukaryotic cells rely on the bidirectional transport of essential substances between the nucleus and cytoplasm. Molecules critical for protein synthesis, such as tRNAs, mRNAs, and pre-ribosomal subunits, are exported from the nucleus to the cytoplasm. In contrast, nuclear proteins, including histones and transcription factors, are imported into the nucleus. This nucleoplasmic transport is mediated by the NPC [ 28 ] . Recent studies have highlighted the relationship between the NPC complex and autophagy. In autophagy-impaired cells, the NPC mediates the essential proteins in the cytoplasm to be translocated to the nucleus and degraded [ 29 ] . Despite the role of NPC in material transport being well-characterized, its biological functions and relationship with autophagy in plant pathogenic fungi remain poorly understood. In this study, we identified a novel nuclear basket nucleoporin, MoNup50, in M. oryzae and investigated its biological functions. Our data revealed that MoNup50 acts as a negative regulator of autophagy. Phenotypic analysis of the Δ Monup50 mutant further demonstrated that MoNup50 plays an essential role in the conidia and appressoria development, as well as pathogenicity in M. oryzae (Fig. 11 ). Phenotypic analysis of the Δ Monup50 mutant revealed that MoNup50 is involved in multiple aspects of fungal development and pathogenicity, including vegetative hyphal growth, spore production, septum formation in conidia, appressorium formation, turgor pressure generation, and the transport and degradation of glycogen and lipid droplets within the appressorium. In M. oryzae , glycogen and lipid droplets are transported to appressoria, where they are metabolized to produce glycerol. This glycerol accumulates in the appressorium, generating turgor pressure that facilitates the formation and penetration of infection pegs into the host plant cuticle, leading to severe infection lesions [ 30 , 31 ] . In this study, the deletion of MoNUP50 impaired the transport and degradation of glycogen and lipid droplets, resulting in insufficient turgor pressure in the Δ Monup50 mutant appressoria. This, in turn, weakened the ability of infectious hyphae to penetrate and expand within the host tissues. In addition to the impact on appressorium function, the integrity of the fungal cell wall is crucial for maintaining turgor pressure in the appressorium. The fungal cell wall is a dynamic structure that remodels during fungal development, plant infection, and in response to external stresses [ 32 ] . Here, we observed impaired cell wall integrity in the Δ Monup50 mutant. Growth inhibition assays in the presence of cell wall stressors such as CFW, SDS, and CR showed a significantly higher inhibition rate in the Δ Monup50 mutant compared to the Guy11 and complemented strains. Furthermore, the Mps1 phosphorylation level was abnormally elevated in the Δ Monup50 mutant under nutrient-rich and CR-induced cell wall stress conditions. The Mps1 MAPK signaling pathway is known to regulate cell wall integrity, and these results further suggest that MoNup50 is essential for maintaining proper cell wall function. The combined defects in appressorium function and cell wall integrity are likely to contribute to the reduced pathogenicity in the Δ Monup50 mutant. Autophagy is a critical process in the fungal pathogenicity. Over the past decade, numerous autophagy-related proteins have been identified in M. oryzae , including MoAtg1-MoAtg10, MoAtg12, MoAtg14, MoAtg15, MoAtg16, and MoAtg18. Deletion of any of these key autophagy genes results in the complete loss of pathogenicity [ 4 ] . Among these, MoAtg8 serves as a key autophagy marker, with the lipidation of MoAtg8 and the degradation of GFP-MoAtg8 being important indicators of autophagic flux [ 33 ] . In our study, we found that the Δ Monup50 mutant exhibited increased lipidated MoAtg8 accumulation and accelerated GFP-MoAtg8 degradation compared to the Guy11. These results suggest that MoNup50 negatively regulates autophagy in M. oryzae , with its deletion resulting in elevated autophagic activity under nutrient-rich and nitrogen-starved conditions. Yeast two-hybrid, GST pull-down, and BiFC assays revealed that MoNup50 interacts with MoAtg7 in vivo and in vitro , suggesting that MoNup50 regulates autophagy by directly affecting the activity of this key autophagy protein. Atg7 is involved in two ubiquitin-like conjugation pathways, including the conjugation of Atg12-Atg5 and Atg8 to the autophagosome membrane [ 25 , 26 ] . Atg8, a ubiquitin-like protein, binds to phosphatidylethanolamine (PE) in a process similar to how ubiquitin binds to target proteins [ 34 ] . The lipidation of Atg8 to PE requires the E1 activating enzyme Atg7, the E2 conjugating enzyme Atg3, and the E3 complex Atg12-Atg5. In this study, we demonstrated that MoNup50 interacts with MoAtg7 and regulates autophagy through this interaction. The Δ Monup50 mutant exhibited increased MoAtg8-PE accumulation, further suggesting that MoNup50 modulates autophagy by influencing the lipidation of MoAtg8 [ 25 ] . In mammalian cardiomyocytes, the overexpression of ATG7 induces autophagy [ 35 ] , while ATG7 deficiency in mice leads to cellular dysfunction and cell cycle arrest in starved mouse embryonic fibroblasts [ 36 ] . In Nicotiana benthamiana , overexpression of ATG7 induces autophagy in plant cells, and Atg7 interacts with several autophagy-related proteins, including Atg3, Atg10, and Atg8 [ 37 ] . Interestingly, Atg8 specifically interacts with the C-terminus of Atg7 [ 38 ] . In M. oryzae , MoAtg7 is critical for pathogenicity, and its deletion results in the complete loss of virulence [ 39 ] . These studies highlight the central role of Atg7 in autophagy regulation, particularly in the conjugation of Atg8 to PE, which is essential for autophagosome membrane expansion. Our study further confirms the involvement of MoAtg7 in M. oryzae pathogenicity, with MoNup50 influencing its function by regulating the autophagic process through MoAtg7. Recent studies have shown that post-translational modifications of MoAtg7 influence autophagy and pathogenicity in M. oryzae . For example, Gcn5, a histone acetyltransferase (HAT) that shuttles between the nucleus and cytoplasm negatively regulates autophagy by acetylating Atg7 [ 9 ] . Future studies will explore whether MoNup50 affects the function of MoAtg7 through post-translational modifications, providing deeper insights into the precise mechanisms by which MoNup50 regulates autophagy and pathogenicity. In conclusion, this study elucidates the critical roles of the nuclear basket nucleoporin MoNup50 in both autophagy regulation and pathogenicity in M. oryzae . MoNup50 regulates autophagy homeostasis by interacting with the key autophagy protein MoAtg7, thereby influencing hyphal vegetative growth, sporulation, spore morphology, appressorium formation, appressorium turgor pressure, degradation and transport of appressorium glycogen and lipid droplets, and pathogenicity in M. oryzae . Future research will focus on understanding how MoNup50 modulates MoAtg7 function to further refine our understanding of the molecular mechanisms underlying fungal pathogenicity. Materials and Methods Generation of deletion mutants and complemented strains Gene knockout was performed using Agrobacterium tumefaciens -mediated transformation( At MT). The pKO3A vector, designed by Lu et al., was used to construct the knockout vector. The upstream (1.5 kb) and downstream fragments (1.5 kb) of MoNUP50 were amplified by PCR using specific primers (Table S1 ). The hygromycin resistance gene fragments ( HPH ) were cloned from pCB1003 using specific primers (Table S1 ). The upstream fragment, HPH , and the downstream fragment were then ligated into the linearized vector pKO3A ( Hind III/ Xba I) using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech co.,ltd). The resulting knockout cassettes were introduced into Guy11 Guy11 strain by At MT. Positive transformants exhibiting HPH resistance were selected on media containing hygromycin B (200 µg/mL) and 5-fluoro-2’-deoxyuridine (0.5 µM). Verification of the mutants was conducted using two sets of PCR primers to confirm the deletion of MoNUP50 . To generate the gene-complemented strains (∆ Monup50::MoNUP50 ), the MoNUP50 sequence, along with its native promoter, was fused with the pKD5 vector ( EcoRI and SmalI -linearized) containing the sulfonylurea resistance gene ( SUR ). The gene-complemented plasmids were then introduced into the ∆ Monup50 mutant. Complemented strains (∆ Monup50::MoNUP50 ) were screened using sulfonylurea (100 µg/mL) and confirmed by fluorescence microscopy. Fungal strains, growth conditions and primers The M. oryzae wild-type strain Guy11 was cultured on complete media (CM) at 25°C with a 16/8 h light/dark photoperiod. For hyperosmotic stress and cell wall stress experiment, the Guy11, the ∆ Monup50 mutant, and the complemented strains were inoculated onto CM media supplemented with 1 M Sorbitol, 0.6 M KCl, 0.6 M NaCl, 50 µg/mL Calcofluor White (CFW), 0.005% SDS and 600 µg/mL Congo Red (CR). All experiments were performed in triplicate. The primers used for PCR amplification in this study are listed in Table S1 . Phenotypic analyses For growth and conidiation experiments, Guy11, Δ Monup50 mutant, and complemented strains were inoculated onto quantitative CM medium at 25°C for 8 days. Colony diameters were measured, and spore production was microscopically quantified. For appressorium-related phenotypic analysis, spores (5×10 4 conidia mL − 1 ) of fungal strains were washed with sterile water, and dropped onto a hydrophobic membrane to induce appressorium formation. Appressorium formation and collapse were monitored and counted. During appressorium formation, glycogen transport was monitored by staining with KI/I 2 solution, while lipid droplet degradation was observed using BODIPY staining. For pathogenicity experiments, mycelial plugs and spore suspensions (5×10 4 conidia mL − 1 ) of fungal strains were inoculated onto detached barley and rice leaves. Disease progression was observed and photographed 4–5 days post-inoculation. Each experiment was repeated three times. Yeast two-hybrid assays To perform yeast two-hybrid assays, the cDNA of MoNUP50 was cloned into the bait vector pGBKT7. The prey vector was constructed by inserting MST50 cDNA into pGADT7. The primers used are listed in Table S1 . Both vectors were co-transformed into the yeast strain Y2HGold, following the manufacturer’s protocol for the Matchmaker Gal4 Two-Hybrid System 3 (Clontech, USA). In vitro GST pull-down assays For the in vitro GST pull-down assays, the cDNA of MoNUP50 was cloned into the Sal1/ Hind III site of pET21a vector to generate the His-MoNup50 plasmid. Similarly, the cDNA of MoATG7 was inserted into the EcoR1 site of the pGEX4T-2 vector (GE Health care Life Science) to generate the GST-MoAtg7 plasmid. Both plasmids were transformed into Escherichia coli strain BL21 (DE3) cells. These cells were harvested and resuspended in a lysis buffer containing10 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 150 mM NaCl and 0.5 mM EDTA. After cell lysis, extracted proteins were analyzed by SDS–PAGE and stained with Coomassie blue. Western blotting was performed to confirm the expression of the target proteins. The GST or GST-MoAtg7 supernatants were incubated with 50 µL glutathione agarose beads (Invitrogen) 4°C for 2 h. Subsequently, His-MoNup50 bacterial lysate were added and incubated at 4°C for 1 h. The eluted proteins were then detected by western blotting using anti-GST and anti-His antibodies (Hua An). Bimolecular fluorescence complementation (BiFC) assay The coding sequences of MoNUP50 and MoATG7 were retrieved from the NCBI database, and their coding regions were analyzed using the EnsemblFungi database to determine the positions of their start and stop codons. Amplified MoNUP50 and MoATG7 fragments were then cloned into the pKD5 and pKD2 vectors, respectively. The resulting MoNup50-YFPC and YFPN-MoAtg7 constructs were co-transformed into the Guy11 via At MT. The positive transformants were selected on plates containing sulfonylurea (100 µg/mL) and hygromycin B (200 µg/mL). YFP signals were detected using a Zeiss LSM880 confocal microscope (Zeiss LSM880). Phosphorylation analysis To measure Mps1 phosphorylation levels, total proteins were extracted from Guy11 and Δ Monup50 mutant using the TCA-acetone method. Protein concentrations were determined using an Enhanced BCA Protein Assay Kit (Beyotime). Phosphorylation of Mps1 was analyzed by western blotting using anti-phospho-p44/42 MAPK antibody and anti-ERK1/2 MAPK antibody (Cell Signaling Technology). Similarly, Osm1 phosphorylation levels were detected using anti-MAPK14 (T180/Y182) antibody and anti-MAPK14 antibody (Cell Signaling Technology). Autophagy assays Autophagic activity was assessed using a GFP-MoAtg8 fusion protein with its native promoter, which was transformed into Guy11 and the Δ Monup50 mutant via At MT method. Transformants expressing GFP-MoAtg8 were analyzed by fluorescence microscopy. The degradation of autophagosome was monitored in both mycelia and conidia using fluorescence microscopy. Furthermore, GFP-MoAtg8 degradation was assessed by western blotting, and the levels of MoAtg8-PE in Guy11 and Δ Monup50 mutants were measured by western blotting after starvation for 0 h, 1 h, and 2 h. Data were analyzed using ImageJ software. Declarations Funding declaration This study was supported by the National Key Research and Development Program of China (2023YFD1400200), the National Natural Science Foundation of China (32300169), and the Key Research and Development Project in Zhejiang Province (2023C02018). Data availability declaration Datasets used or analyzed during the current study are available in the manuscript and supplementary material. Ethics approval and consent to participate No ethics approval or consent was required since this study did not include patient samples, human subjects, or animal experiments. Consent for publication No ethics approval or consent was required since this study did not include patient samples, human subjects, or animal experiments. Competing interests The authors declare no competing interests. Authors’ contributions Ying-Ying Cai: Investigation, Data curation, Methodology, Writing-original draft. Xue-Ming Zhu, Muhammad Noman, Jing Wang: Investigation, Methodology. Zhong-Na Hao, Yan-Li Wang, Lin Li: Data curation, Methodology. Jian-Ping Lu, Xiao-Hong Liu: Investigation, Writing-review & editing, Methodology. Jiao-Yu Wang, Fu-Cheng Lin: Conceptualization, Writing-review & editing, Funding acquisition, Supervision. All authors reviewed the manuscript. Acknowledgments We are grateful to all individuals who contributed to this study. References Chen X, Selvaraj P, Lin LL, Fang WQ, Wu CX, Yang P, et al. Rab7/Retromer-based endolysosomal trafficking is essential for proper host invasion in rice blast. New Phytol. 2023;239(4):1384–403. Zhang F, Wang M, Wang GL, Ning YS, Wang RY. Insights into metabolite biosynthesis and regulation in rice immune signaling. Trends Microbiol. 2023;31(3):225–8. Ryder LS, Talbot NJ. 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Supplementary Files supplementaryfile.zip Supplementary.docx Cite Share Download PDF Status: Published Journal Publication published 29 May, 2025 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Revision requested 01 Feb, 2025 Reviewers agreed at journal 02 Jan, 2025 Reviews received at journal 29 Dec, 2024 Reviewers agreed at journal 21 Dec, 2024 Reviewers invited by journal 21 Dec, 2024 Editor assigned by journal 17 Dec, 2024 Submission checks completed at journal 17 Dec, 2024 First submitted to journal 05 Dec, 2024 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-5584909","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":391359571,"identity":"7ad12052-893e-4be4-b2db-67c6d747ce1f","order_by":0,"name":"Ying-Ying Cai","email":"","orcid":"","institution":"Zhejiang Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Ying-Ying","middleName":"","lastName":"Cai","suffix":""},{"id":391359572,"identity":"171ed42a-d1af-49fb-aefd-099dd9790d6a","order_by":1,"name":"Xue-Ming Zhu","email":"","orcid":"","institution":"Zhejiang Academy of Agricultural 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08:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5584909/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5584909/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-025-02219-7","type":"published","date":"2025-05-29T15:57:34+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71855556,"identity":"a60d78f3-1451-4b6f-9c2b-58106f4bbef6","added_by":"auto","created_at":"2024-12-19 08:08:31","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1969328,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 localizes to the nuclear envelope in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. oryzae.\u003c/strong\u003e\u003c/em\u003e (A) Localization of MoNup50 in the hyphae, conidia and appressorium of \u003cem\u003eM. oryzae\u003c/em\u003e. Scale bar, 10 μm. (B) The fluorescence intensity of GFP-MoNup50 and mCherry-MoH\u003csub\u003e2\u003c/sub\u003eB was measured by ImageJ software.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/790a941537a30f05a72ed7df.png"},{"id":71855507,"identity":"56088b16-9ca5-4ed1-9918-625ffbf72030","added_by":"auto","created_at":"2024-12-19 08:08:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":4527492,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 regulates hyphal growth, spore production, and spore morphology. \u003c/strong\u003e(A) Hyphalgrowth of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. Scale bar, 1 cm. (B) Conidia and conidiophores of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. Scale bar, 50 μm. (C) Conidia morphology of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50.\u003c/em\u003e Scale bar, 10 μm.\u003cem\u003e \u003c/em\u003e(D) Count and analyze the colony growth diameter of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. (E) Statistical analysis of conidiation in Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. (F) Statistical analysis of the conidial septum number of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. The data were analyzed by unpaired two-tailed Student’s t-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003e p\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/a66b59b1ae8e8ddcb5404a6b.png"},{"id":71855541,"identity":"415fac3d-1633-495b-8bab-2222a5a91e95","added_by":"auto","created_at":"2024-12-19 08:08:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4598996,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 regulates appressorium formation and appressorium turgor pressure. \u003c/strong\u003e(A) and (B) appressorium formation and appressorium turgor in Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. Scale bar, 10 μm. (C) and (D) The appressorium formation rates and appressorium collapse rates of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e were analyzed, respectively. The data wereanalyzed by unpaired two-tailed Student’s t-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/0e012d7c4e4490414afabb46.png"},{"id":71856282,"identity":"cf22c603-e892-4e7c-a779-f9c91cb765eb","added_by":"auto","created_at":"2024-12-19 08:16:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":10349728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 is involved in infection and pathogenicity. \u003c/strong\u003e(A) Disease spots of isolated barley leaves inoculated with mycelial plugs of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. (B) Disease symptoms on isolated barley leaves inoculated conidial suspensions of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. (C) Rice seedlings inoculated spore suspensions of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. (D) Penetration assays in isolated barley leaves were separately monitored at different time points. Scale bar, 20 μm. (E) The area occupied by disease spots per 5 cm of rice leaves was counted. (F) Analysis of the number of different invasive hyphae types of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. The data were analyzed by unpaired two-tailed Student’s t-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/b66ac43d8adb2bbb027dc60f.png"},{"id":71855562,"identity":"001f263e-3e7a-4082-8576-801ffc0ab78e","added_by":"auto","created_at":"2024-12-19 08:08:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6112605,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 impairs glycogen and lipid droplet metabolism in appressoria. \u003c/strong\u003e(A) Glycogen distribution in conidia and appressorium of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. Scale bar, 10 μm. (B) and (C) The percentage of conidia and appressoria containing glycogen in Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e, respectively.(D) Lipid droplets distribution in conidia and appressorium of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e. Scale bar, 10 μm. (E) and (F) The percentage of conidia and appressoria containing lipid droplets in Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e, respectively.The data were analyzed by unpaired two-tailed Student’s t-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/c6ec4008a9d912f9f4ea0975.png"},{"id":71855603,"identity":"36c2b2c0-aa7f-45e2-9966-a12a7df98eba","added_by":"auto","created_at":"2024-12-19 08:08:35","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2695196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 regulates the phosphorylation of Osm1 in response to hyperosmotic stresses. \u003c/strong\u003e(A). Colonies of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50 \u003c/em\u003eon CM supplemented with 1 M Sorbitol, 0.6 M KCl and 0.6 M NaCl. (B) Statistical analysis of growth inhibition rates of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e in plates with different hyperosmotic stress factors. (C) The phosphorylation level of Osm1 in Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e mutants. (D) Statistical analysis of the phosphorylated level of Osm1 in Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e. The data were analyzed by unpaired two-tailed Student’s t-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/09e59a8548572be60f4ebca3.png"},{"id":71855527,"identity":"5251bb94-9c5a-4929-967b-8909d82c83a4","added_by":"auto","created_at":"2024-12-19 08:08:30","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2236627,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 regulates the Mps1 phosphorylation level in response to cell wall stress.\u003c/strong\u003e (A) Colonies of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e on CM with 50 μg/mL\u003csup\u003e \u003c/sup\u003eCalcofluor White (CFW), 0.005% SDS and 600 μg/mL\u003csup\u003e \u003c/sup\u003eCongo Red (CR). (B) The phosphorylation level of Mps1 in Guy11 and Δ\u003cem\u003eMonup50 \u003c/em\u003eon 600 μg/mL CR. (C) Statistical analysis of growth inhibition rates of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e in plates with different cell wall stress factors. (D) Statistical analysis of the relative phosphorylation level of Mps1 in Guy11 and Δ\u003cem\u003eMonup50.\u003c/em\u003e The data were analyzed by unpaired two-tailed Student’s t-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001).\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/92ce26bce1a260b420ac43b8.png"},{"id":71855552,"identity":"d8a951f2-83c4-4e25-981b-a2ed790b4419","added_by":"auto","created_at":"2024-12-19 08:08:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":3766610,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 negatively regulates autophagy.\u003c/strong\u003e (A) Growth of the Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e on CM with 100 ng/mL rapamycin. (B) The relative growth inhibition rates of Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e, and ∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e on CM supplemented with 100 ng/mL rapamycin were analyzed. The data were analyzed by unpaired two-tailed Student’s t-test (*\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). (C) The localization of GFP-MoAtg8 in the hyphae of Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e. Scale bar, 10 μm. (D) The localization of GFP-MoAtg8 in the conidia of Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e. Scale bar, 10 μm. (E) The breakdown of GFP-MoAtg8 of Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/d16dc19b896c7c52fd73c152.png"},{"id":71855542,"identity":"16646e1f-695a-4f63-ae46-90e1fa0a9bd0","added_by":"auto","created_at":"2024-12-19 08:08:31","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":2778158,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 interacts with the key autophagy protein MoAtg7 \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e.(A) Yeast two-hybrid analysis to examine the interaction between MoNup50 and MoAtg7 \u003cem\u003ein vivo\u003c/em\u003e. (B) GST-pull down assay for detecting the interaction between MoNup50 and MoAtg7 \u003cem\u003ein vitro\u003c/em\u003e. (C) BiFC assay to detect the interaction between MoNup50 and MoAtg7 \u003cem\u003ein vivo\u003c/em\u003e. Scale bar, 10 μm.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/02125e9e67c4d1059ec3ed99.png"},{"id":71856291,"identity":"4bf9ae02-0fe5-4d5c-9fbf-9daed0f1381e","added_by":"auto","created_at":"2024-12-19 08:16:34","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":694448,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMoNup50 promotes the MoAtg8-PE synthesis.\u003c/strong\u003e (A)Expression level analysis of MoAtg7 by RT-PCR in the Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e. (B)The MoAtg8/MoAtg8-PE turnover in the Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/4b37fff17b089b3f4790cb43.png"},{"id":71855516,"identity":"5e4ffe49-9a24-4af9-a241-2c21be754e31","added_by":"auto","created_at":"2024-12-19 08:08:29","extension":"jpeg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":168230,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModel of MoNup50 regulating the synthesis of MoAtg8-PE through its interaction with the key autophagy protein MoAtg7, thereby affecting pathogenicity and autophagy in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eM. oryzae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image11.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/544553d2ce57c945d09f9cdc.jpeg"},{"id":83783560,"identity":"147a6795-e159-4d01-b8e3-465a4c332206","added_by":"auto","created_at":"2025-06-02 16:11:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36647794,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/3c9f09b6-b4e5-4dcf-8ea1-d15e8f772237.pdf"},{"id":71855559,"identity":"aa5960f4-3d3c-4327-9392-78c20ffdbebf","added_by":"auto","created_at":"2024-12-19 08:08:31","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":5668886,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.zip","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/80109dc8f3394f8542c7fc46.zip"},{"id":71855544,"identity":"80b79a13-9cac-4440-8b67-fa10a6a91d49","added_by":"auto","created_at":"2024-12-19 08:08:31","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1042163,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-5584909/v1/41526d8a53fde6cb72643f7d.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Nuclear basket nucleoporin MoNup50 is essential for fungal development, pathogenicity, and autophagy in Magnaporthe oryzae","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRice blast, caused by the fungal pathogen \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e, is one of the most devastating rice diseases, often referred to as the \u0026ldquo;cancer of rice\u0026rdquo;. It seriously affects global food production and security\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. \u003cem\u003eM. oryzae\u003c/em\u003e primarily infects rice through conidia, which are dispersed by wind and rain. Upon landing on the host leaf surface, the conidium germinates to form an infection structure called an appressorium\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. During appressorium maturation, glycogen and lipid droplets in the conidia translocate to the appressoria. Subsequently, these glycogen and lipid droplets are gradually degraded in the appressoria, leading to the accumulation of substantial glycerol and turgor pressure as high as 8.0 MPa\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. This turgor pressure drives the formation of penetration pegs, enabling the fungus to breach the host leaf surface and establish infection\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAutophagy is a conserved cellular process in which autophagosomes engulf damaged macromolecules and organelles, transporting them to vacuoles or lysosomes for degradation\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. This process maintains cellular homeostasis by recycling macromolecules to replenish energy and material\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. During appressorium maturation, conidia undergo autophagy-dependent nuclear degradation, leading to conidial cell death and recycling of conidial contents into appressorium to facilitate turgor pressure generation\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. Autophagy is regulated by a series of autophagy-related genes (\u003cem\u003eATG\u003c/em\u003es), including \u003cem\u003eATG7\u003c/em\u003e, which regulates autophagy by facilitating the formation of autophagosomes through the Atg8-PE synthesis pathway\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eNuclear pore complexes (NPCs) are large protein assemblies embedded in the nuclear envelope, mediating the transport of biomolecules between the cytoplasm and nucleus\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. NPCs consist of seven substructures formed by approximately 30 different nucleoporins (Nups), playing a particularly dynamic structural and functional role. In \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e, Nup2/Nup50 regulates nuclear transport and interacts with chromatin to influence gene expression\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. In filamentous fungi, Nup2/Nup50 is critical for viability and stress response\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Nup2/Nup50 also interacts with active genes in the nucleoplasm, participates in gene regulation, and controls chromatin epigenetic states by preventing the spread of repressive marks\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. For example, in \u003cem\u003eAspergillus nidulans\u003c/em\u003e, Nup2/Nup50 is critical for the cell viability\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Similarly, Nup2/Nup50 has been reported to be involved in growth and development, stress response, pathogenicity, and DON toxin synthesis in \u003cem\u003eFusarium graminearum\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRecent studies linked the NPC complex to autophagy regulation. In \u003cem\u003eS. cerevisiae\u003c/em\u003e, TORC1 (Tor kinase complex 1) inhibition triggers autophagic degradation of NPC complex and Nups, mediated by selective autophagic receptors such as Atg8\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. In mammals, the nuclear basket Nup, Tpr, regulates autophagy by modulating the export of mRNAs and interacting with autophagy factors\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite extensive studies in yeast and other organisms, the role of Nup50/Nup2 in \u003cem\u003eM. oryzae\u003c/em\u003e, especially in autophagy, remains underexplored. In this study, we characterized MoNup50, a nuclear pore-associated protein, essential for hyphal growth, sporulation, appressorium formation, and pathogenicity. MoNup50 also responds to osmotic and cell wall stress by regulating the Osm1 and Mps1 MAPK signaling pathways. Notably, MoNup50 interacts with the key autophagy protein MoAtg7 to regulate autophagy, and deletion of \u003cem\u003eMoNUP50\u003c/em\u003e resulted in abnormal autophagic activity.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIdentification and structural analysis of MoNup50\u003c/h2\u003e \u003cp\u003eStructural domain analysis revealed that MoNup50 contains the conserved nuclear pore complex protein domain, the FG-repeat region, and the Ran-binding domain (Figure. S1A). This suggests a high degree of conservation of Nup50 across species, including \u003cem\u003eM. oryzae\u003c/em\u003e. Structural predictions of the Nup50 protein indicated similar α-helix structures and disordered FG sequences across different organisms, including yeast, pathogenic fungi, and mammals (Figure. S1B). Phylogenetic analysis of Nup50 from various organisms showed that MoNup50 of \u003cem\u003eM. oryzae\u003c/em\u003e is more closely related to that of \u003cem\u003eF. graminearum\u003c/em\u003e, than to \u003cem\u003eS. cerevisiae\u003c/em\u003e, \u003cem\u003eSchizosaccharomyces pombe\u003c/em\u003e, and mammals (Figure. S1C).\u003c/p\u003e \u003cp\u003e \u003cb\u003eMoNup50 localizes to the nuclear envelope in\u003c/b\u003e \u003cb\u003eM. oryzae\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo determine the subcellular localization of MoNup50 in \u003cem\u003eM. oryzae\u003c/em\u003e, we constructed a MoNup50-GFP fusion vector and transformed it into Δ\u003cem\u003eMonup50\u003c/em\u003e (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The observation under fluorescence microscopy showed that MoNup50-GFP localized around the nucleus. Co-localization of MoNup50-GFP at different developmental stages (hyphae, conidia, and appressoria) with the nuclear marker protein MoH\u003csub\u003e2\u003c/sub\u003eB-mCherry showed that MoNup50-GFP formed a punctate structure around the nucleus in hyphae and a ring-like structure in conidia and mature appressoria, suggesting MoNup50 localizes at the nuclear envelope (Figs.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMoNup50 regulates hyphal growth, spore production, and spore morphology\u003c/h3\u003e\n\u003cp\u003eTo assess the biological function of MoNup50, we created a \u003cem\u003eMoNUP50\u003c/em\u003e deletion strain (Δ\u003cem\u003eMonup50\u003c/em\u003e) and a complemented strain (Δ\u003cem\u003eMonup50\u003c/em\u003e::\u003cem\u003eMoNUP50\u003c/em\u003e) (Figure S2). The Δ\u003cem\u003eMonup50\u003c/em\u003e mutant showed a 41% reduction in colony growth diameter compared to the wild-type (Guy11) and complemented strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Sporulation assays revealed that the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant failed to produce conidia after 24 h, with sporulation reduced by 98% compared to the Guy11 and complemented strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Additionally, the conidia from the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant displayed abnormal morphology, with over 85% showing either no septa or a single septum, whereas more than 90% of Guy11 conidia exhibited two septa (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e2\u003c/span\u003eF). These results indicate that MoNup50 is essential for vegetative growth, spore morphology, and sporulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMoNup50 regulates appressorium formation and appressorium turgor pressure\u003c/h3\u003e\n\u003cp\u003eAt 4, 8, and 12 hours post-inoculation (hpi), the spores of Δ\u003cem\u003eMonup50\u003c/em\u003e mutant exhibited significantly lower appressorium formation rates compared to the Guy11 and complemented strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). 9.70% of the conidia in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant formed appressoria at 4 hpi, compared to over 50% in the Guy11 and complemented strains. At 24 hpi, however, appressorium formation rates were similar between the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant and the Guy11. When submerged in a highly concentrated glycerol solution, the collapse rate of appressoria in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant was significantly higher than in the Guy11 and complemented strains, indicating the mutant\u0026rsquo;s appressoria has lower turgor pressure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings suggest that MoNup50 is crucial for appressorium formation and turgor pressure generation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eMoNup50 is involved in infection and pathogenicity\u003c/h3\u003e\n\u003cp\u003eTo examine the role of MoNup50 in pathogenicity, we first inoculated the Δ\u003cem\u003eMonup50\u003c/em\u003e, Guy11, and complemented strains onto detached barley leaves. At 4 days post-inoculation (dpi), the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant showed few tiny infection lesions, while the Guy11 and complemented strains caused typical coalescent lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similarly, spore suspensions were inoculated onto barley leaves producing only small and restricted lesions by the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant, while the Guy11 and complemented strains caused large and coalescent lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In rice seedling infection assays, the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant caused significantly fewer lesions (lesion area 11.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73%) compared to the Guy11 (78.98\u0026thinsp;\u0026plusmn;\u0026thinsp;3.26%) and complemented strains (77.32\u0026thinsp;\u0026plusmn;\u0026thinsp;2.22%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of barley leaf infection experiments at 36 and 48 hpi revealed that Δ\u003cem\u003eMonup50\u003c/em\u003e has defects in plant penetration and invasive growth. The infection hyphae (IH) were classified into three types, Type I (no IH), and Type II (IH in one cell), Type III (IH penetrating adjacent cell). At 48 hpi, the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant predominantly formed Type I and Type II infection hyphae with significantly less production of Type III hyphae (~\u0026thinsp;16%), whereas the Guy11 and complemented strains formed Type III hyphae (~\u0026thinsp;90%) capable of spreading to adjacent cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eD and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). These results indicate that MoNup50 is crucial for full infection and pathogenicity.\u003c/p\u003e\n\u003ch3\u003eMoNup50 affects stress tolerance and signal transduction pathways\u003c/h3\u003e\n\u003cp\u003eTo investigate the role of MoNup50 in environmental stresses, we conducted sensitivity assays in response to osmotic stressors (sorbitol, NaCl, and KCl) and cell wall stressors (CFW, SDS, and CR). We found that the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant exhibited increased sensitivity to ionic hyperosmotic stress (NaCl and KCl) and cell wall stress (CFW, SDS, and CR), but no significant change in response to non-ionic hyperosmotic stress (Sorbitol) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, S6A and S6C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe Osm1 MAPK and Mps1 MAPK signaling pathways are central pathways to hyperosmotic and cell wall stress responses in \u003cem\u003eM. oryzae\u003c/em\u003e\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. We examined the phosphorylation levels of Osm1 and Mps1 in the Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e mutant. Phosphorylation levels of Osm1 and Mps1 were higher in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant than in the Guy11 under normal and NaCl- or CR-induced conditions, indicating that MoNup50 modulates these signaling pathways during the stress response (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e5\u003c/span\u003eD, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These findings suggest that MoNup50 responds to hyperosmotic stress and cell wall stress by regulating the Osm1 and Mps1 MAPK signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eMoNup50 affects the transport and degradation of glycogen and lipid droplet\u003c/h2\u003e \u003cp\u003eIn \u003cem\u003eM. oryzae\u003c/em\u003e, glycogen and lipid droplets in conidia are degraded and transported to the appressorium \u003cem\u003evia\u003c/em\u003e the germ tube, leading to the accumulation of glycerol, which generates the necessary turgor pressure for infection\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. To assess the cause of reduced turgor pressure in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant, we examined the transport and degradation of glycogen and lipid droplets during appressorium development in three strains. At the conidial stage, no significant difference in glycogen synthesis was observed between the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant and the Guy11. At 8 hpi, 54.12\u0026thinsp;\u0026plusmn;\u0026thinsp;5.44% conidia of the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant contained glycogen, whereas only 15.51\u0026thinsp;\u0026plusmn;\u0026thinsp;3.38% conidia of the Guy11 contained glycogen. At 16 hpi, 93.67% \u0026plusmn; 0.65% of the glycogen in the Guy11 appressoria had been degraded, in contrast, less than 30% of the glycogen was degraded in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant. These data indicate that MoNup50 regulates glycogen transfer and degradation in the conidia and appressoria of \u003cem\u003eM. oryzae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). Intriguingly, there was no significant difference in the transport of lipid droplets in the conidia between the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant and Guy11. However, lipid droplet degradation in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant appressoria was delayed (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eD, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). These findings suggest that MoNup50 is crucial for the transport of glycogen and the degradation of glycogen and lipid droplets in the appressorium.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMoNup50 negatively regulates autophagy\u003c/h3\u003e\n\u003cp\u003eTor is a key regulator of the autophagy process\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Rapamycin is a specific inhibitor of Tor kinase\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. To investigate the role of MoNup50 in autophagy, we inoculated mycelial plugs of Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e mutant onto plates containing 100 ng/mL rapamycin and assessed their growth. The Δ\u003cem\u003eMonup50\u003c/em\u003e mutant showed stronger sensitivity than Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e::\u003cem\u003eMoNUP50\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). These results suggest that MoNup50 plays a role in regulating autophagy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate whether MoNup50 directly influences autophagy, we introduced an autophagy marker GFP-MoAtg8 into the Guy11 and the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant. We examined the subcellular localization of GFP-MoAtg8 and free GFP (which results from the degradation of GFP-MoAtg8 within vacuoles) using a fluorescence microscope (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Under nutrient-sufficient conditions, the GFP-MoAtg8 was mainly localized in the cytoplasm in the Guy11 hyphae, appearing as bright puncta near the vacuoles. In contrast, in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant hyphae, most of the GFP-MoAtg8 entered the vacuoles, with only a few puncta remaining near the vacuoles in the cytoplasm. At 1 h post-starvation, the Guy11 hyphae exhibited GFP-MoAtg8 puncta near the vacuoles, with additional degradation into free GFP within the vacuoles. However, in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant hyphae, all GFP-MoAtg8 degraded into free GFP diffusely distributed within the vacuoles, with no punctate fluorescence observed in the cytoplasm. These observations suggest that the autophagy level is higher in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant than in the Guy11.\u003c/p\u003e \u003cp\u003eAutophagy is also crucial for conidia germination, during which intracellular proteins, macromolecules, and damaged organelles are degraded for cellular recycling. We assessed the autophagy level in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant at spore germination stages by monitoring GFP-MoAtg8 localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Under nutrient-sufficient conditions, GFP-MoAtg8 in the Guy11 was mainly distributed as punctate fluorescence in the cytoplasm, with minimal diffuse fluorescence of free GFP in the vacuoles, indicating a low autophagy level. In contrast, in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant, only a few punctate GFP-MoAtg8 signals were observed in the cytoplasm, with the majority degrading into free GFP in the vacuoles, indicating a higher autophagy level. At 1\u0026ndash;2 h post-starvation, the Guy11 exhibited a decrease in punctate GFP-MoAtg8 signals and an increase in free GFP signals within vacuoles, indicating an increase in autophagy with prolonged starvation. In contrast, the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant displayed almost no punctate GFP-MoAtg8 in the cytoplasm, with the majority of GFP-MoAtg8 degrading into free GFP and accumulating in vacuoles, reflecting a sustained high level of autophagy.\u003c/p\u003e \u003cp\u003eTo quantitatively analyze autophagic flux, we performed western blotting to detect GFP-MoAtg8 and free GFP protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Under nutrient-rich conditions, a strong band for GFP-MoAtg8 was observed in the Guy11, and free GFP was barely detectable. In contrast, GFP-MoAtg8 and free GFP bands were detected at comparable intensities in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant. Specifically, 3% of GFP-MoAtg8 in the Guy11 degraded to free GFP, while 40% of GFP-MoAtg8 degraded to free GFP in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant. At 1 h post-starvation, the band for free GFP in the Guy11 remained weaker than the GFP-MoAtg8 band, but at 2 h post-starvation, the free GFP band became comparable to the GFP-MoAtg8 band. In contrast, in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant, the free GFP band was stronger than the GFP-MoAtg8 at 1 and 2 h post-starvation. Precisely, 43% and 63% of GFP-MoAtg8 degraded into free GFP in the Guy11 at 1 and 2 h post-starvation, respectively. While 74% and 77% of GFP-MoAtg8 degraded into free GFP in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant, respectively. These results indicate that autophagy levels were significantly higher in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant than in the Guy11 under nutrient-rich and starvation conditions.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMoNup50 interacts with the key autophagy protein MoAtg7\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo further validate the relationship between MoNup50 and autophagy, we screened the interaction between MoNup50 and key autophagy protein, MoAtg7. In yeast two-hybrid assays, we found that co-transforming yeast cells with the MoAtg7-AD plasmid and MoNup50-BD plasmid allowed yeast growth on SD-Leu-Trp-His-Ade plates. However, when the MoAtg7-AD plasmid was co-transformed with the pGBKT7 plasmid or the MoAtg7-AD plasmid was co-transformed with the pGADT7 plasmid into the yeast cells, no growth was observed on SD-Leu-Trp-His-Ade plates, indicating that MoNup50 interacts specifically with MoAtg7 without any self-activation (Fig.\u0026nbsp;9A). \u003cem\u003eIn vitro\u003c/em\u003e GST-pull down assays further confirmed the interaction between MoNup50 and MoAtg7. GST-MoAtg7 could pull down His-MoNup50 from the lysate using GST beads, whereas the negative control (empty GST protein) did not pull down any protein (Fig.\u0026nbsp;9B). These results indicate that MoNup50 and MoAtg7 interact directly \u003cem\u003ein vitro\u003c/em\u003e. Additionally, we used the bimolecular fluorescence complementation (BiFC) method to confirm the interaction between MoNup50 and MoAtg7 \u003cem\u003ein vivo\u003c/em\u003e. The C-terminal fragment of \u003cem\u003eMoNUP50\u003c/em\u003e was fused to the pKD5-YFPC vector, and the N-terminal fragment of \u003cem\u003eMoATG7\u003c/em\u003e was fused to the pKD5-YFPN vector. Both constructs were co-transformed into Guy11. As negative controls, we co-transformed YFPN/MoNup50-YFPC and YFPC/YFPN-MoAtg7 into Guy11. YFP signals were detected in the transformants expressing MoNup50-YFPC/YFPN-MoAtg7, but no fluorescence was observed in the negative controls, confirming that MoNup50 interacts with MoAtg7 \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;9C). These results demonstrate that in \u003cem\u003eM. oryzae\u003c/em\u003e, MoNup50 interacts with MoAtg7 \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e.\u003c/p\u003e\n\u003ch3\u003eMoNup50 promotes the MoAtg8-PE synthesis\u003c/h3\u003e\n\u003cp\u003eTo investigate how MoNup50 affects MoAtg7 and its role in the autophagy process, we assessed the relative expression level of MoAtg7 in the Guy11 and the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant. We found that the expression level of MoAtg7 in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant was ~\u0026thinsp;2-fold higher than that in the Guy11 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003eA), suggesting that Monup50 is involved in the regulation of MoAtg7 expression. In the autophagy induction process of \u003cem\u003eS. cerevisiae\u003c/em\u003e, Atg8 undergoes lipidation, a process catalyzed by a series of enzymes, including Atg4, Atg7, Atg3, and the Atg5-Atg12-Atg16 complex. This results in the formation of Atg8-PE, which is anchored to the autophagosome membrane\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Given the close correlation between MoAtg8-PE and the number of autophagosomes\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, we measured the turnover of endogenous MoAtg8 and MoAtg8-PE in the Guy11 and the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant by western blotting to assess autophagy levels. Results showed that MoAtg8 lipidation levels increased over time in the Guy11 and the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant under starvation conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). Notably, under nutrient-rich and starvation conditions, the MoAtg8-PE bands were more prominent in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant compared to the Guy11. At 2 h post-starvation, the MoAtg8-PE/GAPDH ratio in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant reached 1.55, whereas in the Guy11, the ratio was only 0.56 (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e10\u003c/span\u003eB). These results suggest that MoNup50 negatively regulates the conversion process of MoAtg8 to MoAtg8-PE.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEukaryotic cells rely on the bidirectional transport of essential substances between the nucleus and cytoplasm. Molecules critical for protein synthesis, such as tRNAs, mRNAs, and pre-ribosomal subunits, are exported from the nucleus to the cytoplasm. In contrast,\u003c/p\u003e \u003cp\u003enuclear proteins, including histones and transcription factors, are imported into the nucleus. This nucleoplasmic transport is mediated by the NPC\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Recent studies have highlighted the relationship between the NPC complex and autophagy. In autophagy-impaired cells, the NPC mediates the essential proteins in the cytoplasm to be translocated to the nucleus and degraded\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite the role of NPC in material transport being well-characterized, its biological functions and relationship with autophagy in plant pathogenic fungi remain poorly understood. In this study, we identified a novel nuclear basket nucleoporin, MoNup50, in \u003cem\u003eM. oryzae\u003c/em\u003e and investigated its biological functions. Our data revealed that MoNup50 acts as a negative regulator of autophagy. Phenotypic analysis of the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant further demonstrated that MoNup50 plays an essential role in the conidia and appressoria development, as well as pathogenicity in \u003cem\u003eM. oryzae\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePhenotypic analysis of the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant revealed that MoNup50 is involved in multiple aspects of fungal development and pathogenicity, including vegetative hyphal growth, spore production, septum formation in conidia, appressorium formation, turgor pressure generation, and the transport and degradation of glycogen and lipid droplets within the appressorium. In \u003cem\u003eM. oryzae\u003c/em\u003e, glycogen and lipid droplets are transported to appressoria, where they are metabolized to produce glycerol. This glycerol accumulates in the appressorium, generating turgor pressure that facilitates the formation and penetration of infection pegs into the host plant cuticle, leading to severe infection lesions\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In this study, the deletion of \u003cem\u003eMoNUP50\u003c/em\u003e impaired the transport and degradation of glycogen and lipid droplets, resulting in insufficient turgor pressure in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant appressoria. This, in turn, weakened the ability of infectious hyphae to penetrate and expand within the host tissues. In addition to the impact on appressorium function, the integrity of the fungal cell wall is crucial for maintaining turgor pressure in the appressorium. The fungal cell wall is a dynamic structure that remodels during fungal development, plant infection, and in response to external stresses\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. Here, we observed impaired cell wall integrity in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant. Growth inhibition assays in the presence of cell wall stressors such as CFW, SDS, and CR showed a significantly higher inhibition rate in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant compared to the Guy11 and complemented strains. Furthermore, the Mps1 phosphorylation level was abnormally elevated in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant under nutrient-rich and CR-induced cell wall stress conditions. The Mps1 MAPK signaling pathway is known to regulate cell wall integrity, and these results further suggest that MoNup50 is essential for maintaining proper cell wall function. The combined defects in appressorium function and cell wall integrity are likely to contribute to the reduced pathogenicity in the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant.\u003c/p\u003e \u003cp\u003eAutophagy is a critical process in the fungal pathogenicity. Over the past decade, numerous autophagy-related proteins have been identified in \u003cem\u003eM. oryzae\u003c/em\u003e, including MoAtg1-MoAtg10, MoAtg12, MoAtg14, MoAtg15, MoAtg16, and MoAtg18. Deletion of any of these key autophagy genes results in the complete loss of pathogenicity\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Among these, MoAtg8 serves as a key autophagy marker, with the lipidation of MoAtg8 and the degradation of GFP-MoAtg8 being important indicators of autophagic flux\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. In our study, we found that the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant exhibited increased lipidated MoAtg8 accumulation and accelerated GFP-MoAtg8 degradation compared to the Guy11. These results suggest that MoNup50 negatively regulates autophagy in \u003cem\u003eM. oryzae\u003c/em\u003e, with its deletion resulting in elevated autophagic activity under nutrient-rich and nitrogen-starved conditions. Yeast two-hybrid, GST pull-down, and BiFC assays revealed that MoNup50 interacts with MoAtg7 \u003cem\u003ein vivo and in vitro\u003c/em\u003e, suggesting that MoNup50 regulates autophagy by directly affecting the activity of this key autophagy protein.\u003c/p\u003e \u003cp\u003eAtg7 is involved in two ubiquitin-like conjugation pathways, including the conjugation of Atg12-Atg5 and Atg8 to the autophagosome membrane\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Atg8, a ubiquitin-like protein, binds to phosphatidylethanolamine (PE) in a process similar to how ubiquitin binds to target proteins\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. The lipidation of Atg8 to PE requires the E1 activating enzyme Atg7, the E2 conjugating enzyme Atg3, and the E3 complex Atg12-Atg5. In this study, we demonstrated that MoNup50 interacts with MoAtg7 and regulates autophagy through this interaction. The Δ\u003cem\u003eMonup50\u003c/em\u003e mutant exhibited increased MoAtg8-PE accumulation, further suggesting that MoNup50 modulates autophagy by influencing the lipidation of MoAtg8\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. In mammalian cardiomyocytes, the overexpression of \u003cem\u003eATG7\u003c/em\u003e induces autophagy\u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, while \u003cem\u003eATG7\u003c/em\u003e deficiency in mice leads to cellular dysfunction and cell cycle arrest in starved mouse embryonic fibroblasts\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eNicotiana benthamiana\u003c/em\u003e, overexpression of \u003cem\u003eATG7\u003c/em\u003e induces autophagy in plant cells, and Atg7 interacts with several autophagy-related proteins, including Atg3, Atg10, and Atg8\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. Interestingly, Atg8 specifically interacts with the C-terminus of Atg7\u003csup\u003e[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]\u003c/sup\u003e. In \u003cem\u003eM. oryzae\u003c/em\u003e, MoAtg7 is critical for pathogenicity, and its deletion results in the complete loss of virulence\u003csup\u003e[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]\u003c/sup\u003e. These studies highlight the central role of Atg7 in autophagy regulation, particularly in the conjugation of Atg8 to PE, which is essential for autophagosome membrane expansion. Our study further confirms the involvement of MoAtg7 in \u003cem\u003eM. oryzae\u003c/em\u003e pathogenicity, with MoNup50 influencing its function by regulating the autophagic process through MoAtg7. Recent studies have shown that post-translational modifications of MoAtg7 influence autophagy and pathogenicity in \u003cem\u003eM. oryzae\u003c/em\u003e. For example, Gcn5, a histone acetyltransferase (HAT) that shuttles between the nucleus and cytoplasm negatively regulates autophagy by acetylating Atg7\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Future studies will explore whether MoNup50 affects the function of MoAtg7 through post-translational modifications, providing deeper insights into the precise mechanisms by which MoNup50 regulates autophagy and pathogenicity.\u003c/p\u003e \u003cp\u003eIn conclusion, this study elucidates the critical roles of the nuclear basket nucleoporin MoNup50 in both autophagy regulation and pathogenicity in \u003cem\u003eM. oryzae\u003c/em\u003e. MoNup50 regulates autophagy homeostasis by interacting with the key autophagy protein MoAtg7, thereby influencing hyphal vegetative growth, sporulation, spore morphology, appressorium formation, appressorium turgor pressure, degradation and transport of appressorium glycogen and lipid droplets, and pathogenicity in \u003cem\u003eM. oryzae\u003c/em\u003e. Future research will focus on understanding how MoNup50 modulates MoAtg7 function to further refine our understanding of the molecular mechanisms underlying fungal pathogenicity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eGeneration of deletion mutants and complemented strains\u003c/h2\u003e \u003cp\u003eGene knockout was performed using \u003cem\u003eAgrobacterium tumefaciens\u003c/em\u003e-mediated transformation(\u003cem\u003eAt\u003c/em\u003eMT). The pKO3A vector, designed by Lu et al., was used to construct the knockout vector. The upstream (1.5 kb) and downstream fragments (1.5 kb) of \u003cem\u003eMoNUP50\u003c/em\u003e were amplified by PCR using specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The hygromycin resistance gene fragments (\u003cem\u003eHPH\u003c/em\u003e) were cloned from pCB1003 using specific primers (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The upstream fragment, \u003cem\u003eHPH\u003c/em\u003e, and the downstream fragment were then ligated into the linearized vector pKO3A (\u003cem\u003eHind\u003c/em\u003eIII/\u003cem\u003eXba\u003c/em\u003eI) using Phanta Max Super-Fidelity DNA Polymerase (Vazyme Biotech co.,ltd). The resulting knockout cassettes were introduced into Guy11 Guy11 strain by \u003cem\u003eAt\u003c/em\u003eMT. Positive transformants exhibiting \u003cem\u003eHPH\u003c/em\u003e resistance were selected on media containing hygromycin B (200 \u0026micro;g/mL) and 5-fluoro-2\u0026rsquo;-deoxyuridine (0.5 \u0026micro;M). Verification of the mutants was conducted using two sets of PCR primers to confirm the deletion of \u003cem\u003eMoNUP50\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eTo generate the gene-complemented strains (∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e), the \u003cem\u003eMoNUP50\u003c/em\u003e sequence, along with its native promoter, was fused with the pKD5 vector (\u003cem\u003eEcoRI\u003c/em\u003e and \u003cem\u003eSmalI\u003c/em\u003e-linearized) containing the sulfonylurea resistance gene (\u003cem\u003eSUR\u003c/em\u003e). The gene-complemented plasmids were then introduced into the ∆\u003cem\u003eMonup50\u003c/em\u003e mutant. Complemented strains (∆\u003cem\u003eMonup50::MoNUP50\u003c/em\u003e) were screened using sulfonylurea (100 \u0026micro;g/mL) and confirmed by fluorescence microscopy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFungal strains, growth conditions and primers\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eM. oryzae\u003c/em\u003e wild-type strain Guy11 was cultured on complete media (CM) at 25\u0026deg;C with a 16/8 h light/dark photoperiod. For hyperosmotic stress and cell wall stress experiment, the Guy11, the ∆\u003cem\u003eMonup50\u003c/em\u003e mutant, and the complemented strains were inoculated onto CM media supplemented with 1 M Sorbitol, 0.6 M KCl, 0.6 M NaCl, 50 \u0026micro;g/mL Calcofluor White (CFW), 0.005% SDS and 600 \u0026micro;g/mL Congo Red (CR). All experiments were performed in triplicate. The primers used for PCR amplification in this study are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePhenotypic analyses\u003c/h2\u003e \u003cp\u003eFor growth and conidiation experiments, Guy11, Δ\u003cem\u003eMonup50\u003c/em\u003e mutant, and complemented strains were inoculated onto quantitative CM medium at 25\u0026deg;C for 8 days. Colony diameters were measured, and spore production was microscopically quantified. For appressorium-related phenotypic analysis, spores (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e conidia mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of fungal strains were washed with sterile water, and dropped onto a hydrophobic membrane to induce appressorium formation. Appressorium formation and collapse were monitored and counted. During appressorium formation, glycogen transport was monitored by staining with KI/I\u003csub\u003e2\u003c/sub\u003e solution, while lipid droplet degradation was observed using BODIPY staining. For pathogenicity experiments, mycelial plugs and spore suspensions (5\u0026times;10\u003csup\u003e4\u003c/sup\u003e conidia mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) of fungal strains were inoculated onto detached barley and rice leaves. Disease progression was observed and photographed 4\u0026ndash;5 days post-inoculation. Each experiment was repeated three times.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eYeast two-hybrid assays\u003c/h2\u003e \u003cp\u003eTo perform yeast two-hybrid assays, the cDNA of \u003cem\u003eMoNUP50\u003c/em\u003e was cloned into the bait vector pGBKT7. The prey vector was constructed by inserting \u003cem\u003eMST50\u003c/em\u003e cDNA into pGADT7. The primers used are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. Both vectors were co-transformed into the yeast strain Y2HGold, following the manufacturer\u0026rsquo;s protocol for the Matchmaker Gal4 Two-Hybrid System 3 (Clontech, USA).\u003c/p\u003e \u003cp\u003e \u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003eGST pull-down assays\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFor the in vitro GST pull-down assays, the cDNA of \u003cem\u003eMoNUP50\u003c/em\u003e was cloned into the Sal1/\u003cem\u003eHind\u003c/em\u003eIII site of pET21a vector to generate the His-MoNup50 plasmid. Similarly, the cDNA of \u003cem\u003eMoATG7\u003c/em\u003e was inserted into the EcoR1 site of the pGEX4T-2 vector (GE Health care Life Science) to generate the GST-MoAtg7 plasmid. Both plasmids were transformed into \u003cem\u003eEscherichia coli\u003c/em\u003e strain BL21 (DE3) cells. These cells were harvested and resuspended in a lysis buffer containing10 mM Tris-HCl (pH 7.5), 0.5% Triton X-100, 150 mM NaCl and 0.5 mM EDTA. After cell lysis, extracted proteins were analyzed by SDS\u0026ndash;PAGE and stained with Coomassie blue. Western blotting was performed to confirm the expression of the target proteins. The GST or GST-MoAtg7 supernatants were incubated with 50 \u0026micro;L glutathione agarose beads (Invitrogen) 4\u0026deg;C for 2 h. Subsequently, His-MoNup50 bacterial lysate were added and incubated at 4\u0026deg;C for 1 h. The eluted proteins were then detected by western blotting using anti-GST and anti-His antibodies (Hua An).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eBimolecular fluorescence complementation (BiFC) assay\u003c/h2\u003e \u003cp\u003eThe coding sequences of \u003cem\u003eMoNUP50\u003c/em\u003e and \u003cem\u003eMoATG7\u003c/em\u003e were retrieved from the NCBI database, and their coding regions were analyzed using the EnsemblFungi database to determine the positions of their start and stop codons. Amplified \u003cem\u003eMoNUP50\u003c/em\u003e and \u003cem\u003eMoATG7\u003c/em\u003e fragments were then cloned into the pKD5 and pKD2 vectors, respectively. The resulting MoNup50-YFPC and YFPN-MoAtg7 constructs were co-transformed into the Guy11 \u003cem\u003evia At\u003c/em\u003eMT. The positive transformants were selected on plates containing sulfonylurea (100 \u0026micro;g/mL) and hygromycin B (200 \u0026micro;g/mL). YFP signals were detected using a Zeiss LSM880 confocal microscope (Zeiss LSM880).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003ePhosphorylation analysis\u003c/h2\u003e \u003cp\u003eTo measure Mps1 phosphorylation levels, total proteins were extracted from Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e mutant using the TCA-acetone method. Protein concentrations were determined using an Enhanced BCA Protein Assay Kit (Beyotime). Phosphorylation of Mps1 was analyzed by western blotting using anti-phospho-p44/42 MAPK antibody and anti-ERK1/2 MAPK antibody (Cell Signaling Technology). Similarly, Osm1 phosphorylation levels were detected using anti-MAPK14 (T180/Y182) antibody and anti-MAPK14 antibody (Cell Signaling Technology).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAutophagy assays\u003c/h2\u003e \u003cp\u003eAutophagic activity was assessed using a GFP-MoAtg8 fusion protein with its native promoter, which was transformed into Guy11 and the Δ\u003cem\u003eMonup50\u003c/em\u003e mutant \u003cem\u003evia At\u003c/em\u003eMT method. Transformants expressing GFP-MoAtg8 were analyzed by fluorescence microscopy. The degradation of autophagosome was monitored in both mycelia and conidia using fluorescence microscopy. Furthermore, GFP-MoAtg8 degradation was assessed by western blotting, and the levels of MoAtg8-PE in Guy11 and Δ\u003cem\u003eMonup50\u003c/em\u003e mutants were measured by western blotting after starvation for 0 h, 1 h, and 2 h. Data were analyzed using ImageJ software.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Key Research and Development Program of China (2023YFD1400200), the National Natural Science Foundation of China (32300169), and the Key Research and Development Project in Zhejiang Province (2023C02018).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDatasets used or analyzed during the current study are available in the manuscript and supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethics approval or consent was required since this study did not include patient samples, human subjects, or animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo ethics approval or consent was required since this study did not include patient samples, human subjects, or animal experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYing-Ying Cai: Investigation, Data curation, Methodology, Writing-original draft. Xue-Ming Zhu, Muhammad Noman, Jing Wang: Investigation, Methodology. Zhong-Na Hao, Yan-Li Wang, Lin Li: Data curation, Methodology. Jian-Ping Lu, Xiao-Hong Liu: Investigation, Writing-review \u0026amp; editing, Methodology. Jiao-Yu Wang, Fu-Cheng Lin: Conceptualization, Writing-review \u0026amp; editing, Funding acquisition, Supervision. All authors reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are grateful to all individuals who contributed to this study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eChen X, Selvaraj P, Lin LL, Fang WQ, Wu CX, Yang P, et al. Rab7/Retromer-based endolysosomal trafficking is essential for proper host invasion in rice blast. New Phytol. 2023;239(4):1384\u0026ndash;403.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang F, Wang M, Wang GL, Ning YS, Wang RY. 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Proc Natl Acad Sci USA. 2009;106(37):15967\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e\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":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"rice blast fungus, virulence, Atg7, nuclear pore complex","lastPublishedDoi":"10.21203/rs.3.rs-5584909/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5584909/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAutophagy is crucial for appressorium development and host invasion by phytopathogenic fungi, including \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e. During appressorium maturation, many organelles, such as nuclei, in the conidia need to be degraded through autophagy to be recycled in the appressorium. However, the interplay between autophagy and nuclear membrane systems remains poorly understood. In this study, we functionally characterized MoNup50, a nuclear pore-associated protein. Despite sharing limited sequence identity with human and yeast Nup proteins, MoNup50 contains conserved domains typical of nuclear pore complex proteins. Observation under fluorescence microscopy revealed that MoNup50 localizes to the nuclear membrane in \u003cem\u003eM. oryzae\u003c/em\u003e. Deletion of \u003cem\u003eMoNUP50\u003c/em\u003e resulted in reduced hyphal growth, spore production, appressorium formation, and pathogenicity, while increasing sensitivity to osmotic stress and cell wall disruption. Notably, MoNup50 interacts with the key autophagy protein MoAtg7, which regulates MoAtg8-PE synthesis during autophagy. Moreover, \u003cem\u003eMoNUP50\u003c/em\u003e deletion led to elevated autophagy levels and increased phosphorylation of the MAPKs Osm1 and Mps1. These findings suggest that MoNup50 is involved in appressorium morphogenesis and pathogenicity by modulating autophagy and MAPK pathways, highlighting the critical role of nuclear pore proteins in \u003cem\u003eM. oryzae\u003c/em\u003e pathogenicity and their potential cross-talk with autophagic and MAPK signaling.\u003c/p\u003e","manuscriptTitle":"Nuclear basket nucleoporin MoNup50 is essential for fungal development, pathogenicity, and autophagy in Magnaporthe oryzae","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 08:08:09","doi":"10.21203/rs.3.rs-5584909/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-02-01T16:49:31+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"21606078704571868681577848146895484220","date":"2025-01-02T18:17:16+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-12-29T18:44:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"175239157673060009959749240713341554807","date":"2024-12-21T19:30:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-12-21T18:24:49+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-12-17T11:10:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-12-17T11:07:41+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2024-12-05T08:01:57+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09d7b8af-3db5-4dee-a976-c67edbaaf876","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-06-02T16:08:28+00:00","versionOfRecord":{"articleIdentity":"rs-5584909","link":"https://doi.org/10.1186/s12964-025-02219-7","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2025-05-29 15:57:34","publishedOnDateReadable":"May 29th, 2025"},"versionCreatedAt":"2024-12-19 08:08:09","video":"","vorDoi":"10.1186/s12964-025-02219-7","vorDoiUrl":"https://doi.org/10.1186/s12964-025-02219-7","workflowStages":[]},"version":"v1","identity":"rs-5584909","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5584909","identity":"rs-5584909","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}