Summary: Reactive oxygen species (ROS) induce the synthesis of a myriad of secondary metabolites, including aflatoxins. It raises significant concern as it is a potent environmental contaminant. In Aspergillus flavus ., antioxidant enzymes link ROS stress response with coordinated gene regulation of aflatoxin biosynthesis. In this study, we characterized the function of a core component of the antioxidant enzyme catalase (CTA1) of A . flavus . Firstly, we verified the presence of cta1 corresponding protein (CTA1) by Western blot analysis and mass‐spectrometry based analysis. Then, the functional study revealed that the growth, sporulation and sclerotia formation significantly increased, while aflatoxins production and virulence were decreased in the cta1 deletion mutant as compared with the WT and complementary strains. Furthermore, the absence of the cta1 gene resulted in a significant rise in the intracellular ROS level, which in turn added to the oxidative stress level of cells. A further quantitative proteomics investigation hinted that in vivo , CTA1 might maintain the ROS level to facilitate the aflatoxin synthesis. All in all, the pleiotropic phenotype of A . flavus CTA1 deletion mutant revealed that the antioxidant system plays a crucial role in fungal development, aflatoxins biosynthesis and virulence.

Reversible phosphorylation by protein kinases and phosphatases plays a central role in regulating cellular processes. However, knowledge of the functions of protein phosphatase 2C (PP2C) S/T phosphatases in Aspergillus flavus has been unreported until now. Here, we have identified seven members of the PP2C family of protein phosphatases in A. flavus . Evolutionary and functional analyses indicated that two redundant PP2C phosphatases, Ptc1 and Ptc2, are highly conserved and regulate conidia development, aflatoxin synthesis, seed infection, and autophagic vesicle formation. The cytoplasmic proteins Ptc1 and Ptc2 exhibit nuclear infiltration after DNA damage-induced autophagy. Their degradation is closely linked to autophagy induction. The Asp residue coordinated with Mg ²⁺ is critical for phosphatase Ptc1 and Ptc2 activity, thermal stability, and biofunction in A. flavus . An immunoprecipitation-mass spectrometry proteomic investigation indicated that 133 proteins co-interact with Ptc1 and Ptc2. Among these proteins, phosphoglycerate kinase 1 (PGK1) interacts with Ptc1 and Ptc2 and shows high levels of phosphorylation in Δ ptc1 , Δ ptc2 , and Δ ptc1 / ptc2 mutants. Furthermore, PGK1 S203 phosphorylation levels are associated with aflatoxin synthesis and autophagic vesicle formation. Overall, these findings contribute to our understanding of the roles and mechanisms of PP2C family phosphatases in A. flavus and highlight their importance in various cellular processes. Furthermore, it reveals a new regulation model in A. flavus , where Ptc1 and Ptc2 activate autophagy and aflatoxin synthesis by regulating PGK1. IMPORTANCE: Aspergillus flavus is a model filamentous fungus that can produce aflatoxins when it infects agricultural crops. This study evaluated the protein phosphatase 2C (PP2C) family as a potential drug target with important physiological functions and pathological significance in A. flavus . We found that two redundant PP2C phosphatases, Ptc1 and Ptc2, regulate conidia development, aflatoxin synthesis, autophagic vesicle formation, and seed infection. The target protein phosphoglycerate kinase 1 (PGK1) that interacts with Ptc1 and Ptc2 is essential to regulate metabolism and the autophagy process. Furthermore, Ptc1 and Ptc2 regulate the phosphorylation level of PGK1 S203, which is important for influencing aflatoxin synthesis. Our results provide a potential target for interdicting the toxicity of A. flavus .

Aspergillus flavus (A. flavus), a pathogenic fungus, can produce carcinogenic and toxic aflatoxins that are a serious agricultural and medical threat worldwide. Attempts to decipher the aflatoxin biosynthetic pathway have been hampered by the lack of a high-quality genome annotation for A. flavus. To address this gap, we performed a comprehensive proteogenomic analysis using high-accuracy mass spectrometry data for this pathogen. The resulting high-quality data set confirmed the translation of 8724 previously predicted genes and identified 732 novel proteins, 269 splice variants, 447 single amino acid variants, 188 revised genes. A subset of novel proteins was experimentally validated by RT-PCR and synthetic peptides. Further functional annotation suggested that a number of the identified novel proteins may play roles in aflatoxin biosynthesis and stress responses in A. flavus. This comprehensive strategy also identified a wide range of posttranslational modifications (PTMs), including 3461 modification sites from 1765 proteins. Functional analysis suggested the involvement of these modified proteins in the regulation of cellular metabolic and aflatoxin biosynthetic pathways. Together, we provided a high-quality annotation of A. flavus genome and revealed novel insights into the mechanisms of aflatoxin production and pathogenicity in this pathogen.

Summary: Ras subfamily proteins are molecular switches in signal transduction pathways of many eukaryotes that regulate a variety of cellular processes. Here, the Ras subfamily, encoded by six genes, was identified in Aspergillus flavus : rasA , rasB , rasC , rab‐33 , rheb and rsr1 . The rsr1 deletion mutant (∆ rsr1 ), rheb deletion mutant (∆ rheb ) and double deletion mutant (∆ rheb/rsr1 ) displayed significantly decreased growth and sporulation. Sclerotia formation was significantly decreased for ∆ rheb or ∆ rheb/rsr1 but increased for ∆ rsr1 . Aflatoxin production was significantly increased in ∆ rheb but decreased in ∆ rsr1 and ∆ rheb/rsr1 . We found that rsr1 and rheb are crucial for the pathogenicity of A . flavus . Quantitative proteomics identified 520 differentially expressed proteins (DEPs) for the ∆ rsr1 mutant and 133 DEPs for the ∆ rheb mutant. These DEPs were annotated in multiple biological processes and KEGG pathways in A . flavus . Importantly, we identified the cytokinesis protein SepA in the protein–protein interaction network of rsr1 , and deletion mutants showed that SepA has pleiotropic effects on growth and AF biosynthesis, which may depend on Rsr1 for regulation in A . flavus . Our results indicated that these Ras subfamily proteins exhibited functional redundancy with each other but there were also differences in A . flavus .

Metabolic reprogramming is a hallmark of psoriasis, yet the contribution of lactate metabolism to keratinocyte-mediated immune dysregulation remains undefined. Through integrated bulk and single-cell RNA sequencing, validated by immunofluorescence and metabolic assays, we identified the mitochondrial protein SCO2 as a key pathogenic hub gene upregulated in psoriatic lesions. Functionally, SCO2 overexpression promoted keratinocyte migration and triggered a metabolic shift characterized by mitochondrial pyruvate accumulation and intracellular lactate retention. Single-cell analysis further revealed that SCO2-high keratinocytes establish pathogenic crosstalk with CCR7⁺ dendritic cells via MIF-(CD74 + CD44) interactions, wherein these CCR7⁺ dendritic cells serve as the primary source of IL-23 and co-stimulatory signals (CD80/CD86) to drive robust T cell priming. Our findings highlight SCO2 as a pivotal immunometabolic switch linking keratinocyte metabolism to adaptive immunity. Targeting SCO2 offers a novel strategy to disrupt the keratinocyte-driven recruitment of CCR7⁺ DCs, thereby attenuating the IL-23-mediated inflammatory cascade. Furthermore, SCO2 may serve as a potential biomarker for metabolic dysregulation in psoriatic lesions.