Role of the OTAbZIP Gene in Regulating Growth and OTA Production in Aspergillus westerdijkiae fc-1 Under Osmotic Stress

Role of the OTAbZIP Gene in Regulating Growth and OTA Production in Aspergillus westerdijkiae fc-1 Under Osmotic Stress | 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 Role of the OTAbZIP Gene in Regulating Growth and OTA Production in Aspergillus westerdijkiae fc-1 Under Osmotic Stress Yanling Ma, Muyuan Zhuang, Tanvir Ahmad, Yuhong Yan, Weitian Yuan, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5792788/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Ochratoxins are toxins primarily produced by fungi from the Aspergillus and Penicillium genera. Among these, Ochratoxin A (OTA) is the most toxic and exhibits strong carcinogenic effects. Reducing food contamination by OTA is a critical global challenge. Osmotic pressure is an important environmental factor that regulates various metabolic pathways in Aspergillus westerdijkiae . It particularly affects the HOG pathway, which controls fungal mycelium growth and OTA production. The OTAbZIP gene is a key transcription factor and receptor in A. westerdijkiae . It plays a vital role in responding to osmotic pressure and regulating the expression of OTA biosynthesis genes ( otaA-D ). This study explored the role of OTAbZIP in fungal growth and OTA production under different osmotic conditions using RNA-Seq and RT-qPCR analyses. Results Transcriptomic analysis showed that the OTAbZIP gene influences several pathways. These include DNA replication, sugar metabolism, ribosome function, and arginine and proline metabolism at NaCl concentrations of 0, 20, and 100 g/L. Genes in the high osmolarity glycerol, mitogen-activated protein kinase (MAPK HOG) pathway, such as Hog1 , Gpd1 , Cdc28 , and Ctt1 were affected. The ΔOTAbZIP mutant strain exhibited increased sensitivity to osmotic stress. RT-qPCR results confirmed that OTA biosynthesis gene expression was significantly lower in the ΔOTAbZIP mutant strain than in the wild-type (WT) strain of A. westerdijkiae fc-1. However, the ΔOTAbZIP mutation did not affect the activation of the Hog1 gene. Conclusions This study highlights the role of the OTAbZIP gene in regulating metabolic pathways linked to growth and OTA production in A. westerdijkiae fc-1. These findings enhance our understanding of the gene's functions of A. westerdijkiae fc-1 under various somatic pressures. They also offer insights for developing strategies to control OTA contamination in food and feed. OTA osmotic stress Aspergillus westerdijkiae OTAbZIP gene knockout HOG-MAPK pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Ochratoxin A (OTA) is one of the most prevalent mycotoxins in food and feed. It is a low molecular weight secondary metabolite produced by certain species of Aspergillus and Penicillium [ 1 ]. OTA commonly contaminates cereals, pulses, dried fruits, grapes, tea, and animal feeds. Human and animal exposure to OTA has been associated with acute and chronic toxicity, including carcinogenicity and renal dysfunction [ 2 – 4 ]. OTA contamination primarily occurs through the consumption of naturally contaminated food and feed. In 2016, the International Agency for Research on Cancer (IARC) classified OTA as a Group 2B carcinogen [ 5 ]. Aspergillus westerdijkiae is one of the most important OTA-producing species of Aspergillus and a significant contaminant of food products [ 6 , 7 ]. Recent studies have shown that AwAreA , a nitrogen-responsive transcriptional regulator in A. westerdijkiae , plays a crucial role in regulating nitrogen sources. It significantly impacts the strain's growth and conidial formation [ 8 ]. Under high osmotic stress, ΔAwSakA mutants of A. westerdijkiae exhibited reduced mycelial growth and decreased expression of the biosynthetic genes otaA , otaB , and otaD [ 9 ]. The absence of the transcription factor AwHog1 in A. westerdijkiae also resulted in reduced mycelial growth and reduced OTA production [ 10 ]. Aspergillus species are constantly influenced by various environmental factors, with osmotic pressure being one of the most critical. This factor plays a pivotal role in regulating their mycelial growth and mycotoxin production [ 11 ]. The mitogen-activated protein kinase (MAPK) signaling pathway is widespread and highly conserved in eukaryotic cells. It regulates transcriptional activity and biochemical responses by phosphorylating downstream transcription factors. Additionally, it modulates various enzymes, enabling the cell to adapt to changes in the external environment [ 12 , 13 ]. The Hog1 -MAPK signaling pathway can be activated by extracellular high osmotic stress and plays a crucial role in protecting cells in high osmotic pressure environments [ 14 ]. The OTAbZIP gene encodes a basic leucine zipper ( bZIP ) transcription factor, which is one of the most widely distributed types of transcription factors in eukaryotes. The bZIP transcription factors regulate gene expression by binding to cis-acting elements within promoter sequences upstream of structural genes, playing a critical role in activating or repressing transcription [ 15 – 17 ]. The OTAbZIP gene also controls the expression of the OTA biosynthesis genes [ 15 ]. Osmotic pressure signaling is essential for maintaining cellular homeostasis and development in fungi. The bZIP transcription factors are key players in the regulatory networks of this signaling [ 18 ]. A. westerdijkiae Sarcolamban B ( AwSclB ) gene may have a negative regulatory effect on OTA synthesis through its interaction with OTAbZIP [ 19 ]. The atfA gene of Penicillium marneffei , which encodes a bZIP -type transcription factor, was characterized. The atfA mutant exhibited increased sensitivity to sodium dodecyl sulfate and tert-butyl hydroperoxide compared to wild-type strains. However, it showed no sensitivity to sodium chloride, sorbitol, ultraviolet light and heat stress.[ 20 ]. The bZIP transcription factor atf1 in wood fungi plays a key role in blue light response and MAPK HOG1 downstream signaling [ 21 ]. However, the impact of the OTAbZIP gene on differential gene expression within the mycotoxin-producing metabolic pathway and the HOG pathway of A. westerdijkiae fc-1 strains under varying osmolarity conditions remains unclear. To explore this, a mutant strain of Δ OTAbZIP A. westerdijkiae was constructed to investigate how the OTAbZIP gene influences metabolic pathways and gene expression related to OTA production. The recent advancements in sequencing technology, RNA-Seq has emerged as a cost-effective tool that greatly enhances bioinformatics analysis [ 22 ]. The objectives of this study were to investigate the role of the environmental regulator OTAbZIP gene in A. westerdijkiae fc-1 under different osmotic pressure conditions using transcriptome sequencing (RNA-Seq). Specifically, we aimed to determine how the OTAbZIP gene affects metabolic pathways related to mycelium growth and OTA production, with a focus on the HOG pathway and differentially expressed genes. Additionally, we analyzed the expression of Hog1 and OTA biosynthesis genes, key components of the HOG pathway, through RT-PCR to verify the impact of OTAbZIP on these crucial genes. The results will help to refine the role of the OTAbZIP gene in regulating metabolic pathways related to the growth and virulence production of A. westerdijkiae strains under different osmotic pressure conditions. 2. Results 2.1 Analysis of RNA sequencing results The clean reads were obtained from 18 samples after filtering out low-quality sequences (Table 1 ). All bases met the Q30 criteria, exceeding 91%, and the GC content ranged between 35% and 65%. Finally, a total of 79,305,730 and 302 genes were identified, with an average length of 643.2 bp. These results indicated high sequencing quality, suitable for further analysis. Table 1 Summary of RNA sequencing metrics for WT and ΔOTAbZIP mutant strains under different NaCl concentrations*. Sample Clean reads Error rate (%) Q > 30 (%) GC content (%) WT_0 g/L NaCl 43632895 0.03 92.32 52.89 Δ OTAbZIP _0 g/L NaCl 43858039 0.03 91.57 50.03 WT_20 g/L NaCl 41636893 0.03 92.06 49.32 Δ OTAbZIP _20 g/L NaCl 42754401 0.03 92.16 47.86 WT_100 g/L NaCl 44531399 0.03 92.28 51.80 Δ OTAbZIP _100 g/L NaCl 42993410 0.03 92.15 45.79 *Q > 30 is the percentage of bases with a mass value greater than 30 in the total bases with an error rate < 0.1%; GC is the percentage of G and C bases in the total bases in the number of reads after filtering . 2.2 Differential gene expression 2.2.1. Clustering Analysis Differential genes were compiled from all comparison groups, allowing for cluster analysis of expression patterns across six sample sets. A heatmap was generated to illustrate gene clustering, where color intensity reflects normalized expression values (typically between − 2 and 2). The heatmap enables horizontal comparisons of gene expression across different samples. 2.2.2 Volcano Plot Analysis Volcano plots (Fig. 3 ) visualize the distribution of differential gene expression across various comparisons. The horizontal axis represents the log 2 fold change in gene expression between treated and control groups while the vertical axis indicates the significance of these differences (-log10 adjusted p- value). Up-regulated genes are marked with red dots and down-regulated genes with green dots. In the comparison of A. westerdijkiae WT and mutant Δ OTAbZIP at 0 g/L NaCl, a total of 13,294 differentially expressed genes (DEGs) were identified, including 968 up-regulated and 1,254 down-regulated genes (Fig. 3 A). While under 20 g/L NaCl, a total 13,200 DEGs were observed, with 1,575 up-regulated and 2,033 down-regulated genes (Fig. 3 B). At 100 g/L NaCl, a total of 12,585 DEGs were found, comprising 208 up-regulated and 316 down-regulated genes (Fig. 3 C). 2.3 Differential gene GO function analysis Gene Ontology (GO) analysis categorizes gene functions into biological processes, cellular components, and molecular functions, with results displayed in Fig. 4 (A, B, C). The x-axis indicates the significance level of GO term enrichment, with higher values denoting greater significance. The y-axis lists the GO terms and color coding differentiates between biological processes (BP), cellular components (CC), and molecular functions (MF), with a significance threshold set at padj < 0.05. In the 0 g/L NaCl comparison, significant biological processes included nucleoside metabolism, glycosyl complex metabolism, and DNA replication, with no notable changes in cellular composition. Molecular functions such as oxidoreductase activity and RNA endonuclease activity also showed significance (Fig. 4 A). At 20 g/L NaCl, significant processes included DNA replication and metal ion transport, alongside notable cellular components like the cytoskeleton and endoplasmic reticulum membrane (Fig. 4 B). At 100 g/L NaCl, significant processes related to nucleoside metabolism and carbohydrate derivatives emerged, with notable changes in membrane protein complexes and other cellular components (Fig. 4 C). Differential genes in biological processes such as nucleoside metabolic processes, glycosyl complex metabolic processes and DNA replication were significant in A. westerdijkiae WT compared with the mutant Δ OTAbZIP strain under 0 g/L NaCl. No significant changes in physiological function in terms of cellular composition were produced. Oxidoreductase activity, ribonucleic acid endonuclease activity, methyltransferase activity, and transmembrane transporter protein activity were shown to have significant plots for genes differing in molecular function. Differential genes in biological processes such as DNA replication, metal ion transport, transporter nucleoside metabolism processes, and glycosyl complex metabolism processes were significant in A.westerdijkiae wild-type WT compared with the mutant strain Δ OTAbZIP at 20 g/L NaCl. Differential genes were significant in cellular components such as cytoskeleton, ribosomal outer membrane, endoplasmic reticulum membrane. Differential genes were significant in molecular functions such as metal ions, transmembrane transporter activity, microtubule motility activity, and antioxidant activity Differential genes in biological processes such as nucleoside metabolism processes, glycosyl compound metabolism processes, small molecule metabolism processes containing nucleobases, and carbohydrate derivative metabolism processes were significant in A.westerdijkiae wild-type WT compared with the mutant strain Δ OTAbZIP at 100 g/L NaCl. Differential genes in membrane protein complexes, plasma membranes, cytoplasmic vesicles, and other cellular components were significant. Differential genes in molecular functions such as flavin adenine dinucleotide binding, endonuclease activity, monooxygenase activity, and hydrolase activity have significant plots. 2.4. Functional analysis of the differential gene via KEGG Figure 5 illustrates the KEGG enrichment results, highlighting the 20 most significant pathways. The x-axis reflects the ratio of annotated differential genes to the total number of DEGs, while the y-axis lists the KEGG pathways. The size of the dots indicates the number of genes associated with each pathway, and colors range from red to purple to represent enrichment significance. Key KEGG pathways enriched at 0 g/L NaCl included DNA replication, fructose and mannose metabolism, steroid biosynthesis, pyruvate metabolism, and starch and sucrose metabolism (Fig. 5 A). At 20 g/L NaCl, significant pathways encompass sugar metabolism, ribosomal processes, and arginine and proline metabolism (Fig. 5 B). At 100 g/L NaCl, differential genes were enriched in pathways related to acetate and dicarboxylate metabolism, oxidative phosphorylation, and unsaturated fatty acid biosynthesis (Fig. 5 C). 2.5 HOG-MAPK pathway differential gene analysis The results of the KEGG pathway analysis highlighting the HOG-MAPK pathway are illustrated in Fig. 6 . In this figure, KEGG nodes representing up-regulated genes are denoted in red, while those representing down-regulated genes are marked in green. The shading of the colors corresponds to the significance level of the gene expressions. The mitogen-activated protein kinase (MAPK) signaling pathway is a highly conserved and ubiquitous system in eukaryotic cells, playing a crucial role in cellular signal transduction. Specifically, the Hog1 MAPK pathway is activated by external stimuli, such as high osmotic stress, and is vital for cell survival in hyperosmotic conditions [ 23 ]. Previous studies have demonstrated that the NaCl-induced production of OTA is associated with the phosphorylation status of the HOG MAP kinase [ 24 ]. Under 0g/L NaCl, the comparison between the wild-type (WT) and the mutant ΔOTAbZIP strain revealed a down-regulation of the mitogen-activated protein kinase-encoding gene Hog1 , alongside a significant down-regulation of the glycerol synthetase-encoding gene Gpd1 within the HOG-MAPK pathway (Fig. 6 A). while under 20 g/L NaCl, the analysis showed an up-regulation of the HOG-MAPK pathway, particularly involving the cell cycle protein-dependent serine Cdc28 . Conversely, there was down-regulation observed in the genes encoding Hog1 , the protein kinase Pbs2 , and the catalase gene Ctt1 (Fig. 6 B). At under 100 g/L NaCl, the comparison indicated down-regulation of the Hog1 gene within the HOG-MAPK pathway, consistent with the previous findings under varying NaCl concentrations (Fig. 6 C) 2.6. Differential Gene Protein Interaction Network Protein-protein interactions (PPIs) played important roles in the molecular recognition of pathogens. Figure 7 represented the PPIs network views of the WT strain and mutant ΔOTAbZIP strain under 0, 20, and 100 g/L NaCl conditions, respectively. Each node in the interworking network graph represents a protein, the size of the node is proportional to the degree of this node, and the color gradient of the node from yellow to red corresponds to the value of the aggregation factor from low to high. By mapping the differential gene-protein interactions network, we found that the candidate gene under the 0 g/L NaCl condition was TU AoFC_00282 (Fig. 7 A) and under the 20 g/L NaCl condition, it was TU AoFC_06641 (Fig. 7 B) while under the 100 g/L NaCl condition, it was TU AoFC_09006 and TU AoFC_09007 (Fig. 7 C). 2.7 OTA biosynthetic genes and Hog1 gene expression under different osmolarity conditions The expression levels of OTA biosynthesis genes ( otaA-D ) and the Hog1 gene were analyzed using quantitative reverse transcription PCR (qRT-PCR), as presented in Fig. 8. At 0 g/L NaCl, comparison of the wild-type (WT) strain of A.westerdijkiae fc-1 with the mutant strain Δ OTAbZIP revealed that the transcriptional expression level of the Hog1 gene in the mutant strain increased by 26.02%. In contrast, the expression levels of the OTA biosynthesis genes showed significant decreases: otaA decreased by 3.19-fold, otaB by 2.64-fold, and otaC by 5.51-fold, while the expression of otaD in the Δ OTAbZIP mutant strain increased by 23.65% compared to the WT strain. At 20 g/L NaCl, the comparison again demonstrated an increase in the transcriptional expression of the Hog1 gene in the mutant Δ OTAbZIP , approximately 1.12-fold compared to the WT. However, the OTA biosynthesis genes experienced dramatic decreases: otaA decreased by 8,360-fold, otaB by 24.82-fold, otaC by 160.42-fold, and otaD by 40.58-fold. At 100 g/L NaCl, the expression level of the Hog1 gene in the Δ OTAbZIP mutant strain increased by 40.77% compared to the WT, while the OTA biosynthesis genes again showed substantial down-regulation, with otaA decreasing by 5.34-fold, otaB by 3.77-fold, otaC by 12.12-fold, and otaD by 4.02-fold. Overall, these results indicate that increasing NaCl concentrations significantly affect the transcriptional expression of both the Hog1 gene and the OTA biosynthesis genes, with distinct patterns observed in the wild-type versus mutant strains. 3. Discussion This study used RNA-Seq technology to investigate the differentially expressed genes in WT and Δ OTAbZIP mutant A. westerdijkiae fc-1 strains. These strains were examined under different NaCl concentration conditions. KEGG pathway analysis was also performed to gain a more comprehensive and accurate understanding of how the OTAbZIP gene regulates OTA production under varying osmotic stress conditions [ 25 ]. Notably, the DEGs in both WT and ΔOTAbZIP strains varied significantly under the different NaCl concentrations, indicating a substantial influence of osmotic stress on gene expression [ 26 ]. Möhlmann et al. (2015) demonstrated that nucleotides participate in various cellular processes, including nucleoside monophosphate synthesis and the modification of nucleic acids, which aligns with the transcriptomic analysis [ 27 ]. At 0 g/L NaCl, GO functional analysis revealed significant changes in the biological processes of nucleoside metabolism, glycosyl complex metabolism, and DNA replication in both WT and ΔOTAbZIP mutant strains. These results suggest that the OTAbZIP gene influences cellular reactions by modulating glycosyl complex metabolism, which impacts purine and pyrimidine nucleotide metabolism, including DNA replication. Maintaining nucleotide metabolism balance appears to be essential for the proper development of A. westerdijkiae . In line with this, a recent study by Sellés Vidal et al. (2021) emphasized the role of oxidoreductases in central metabolic pathways [ 28 ]. Interestingly, the lack of significant changes in bio-composition under varying NaCl conditions suggests that the deletion of OTAbZIP did not drastically affect the overall physiological functions of A. westerdijkiae . However, in terms of molecular function, the differential expression of genes involved in oxidoreductase activity, ribonuclease activity, methyltransferase activity, and transmembrane transporter protein activity indicates that the OTAbZIP gene likely regulates several key metabolic pathways. This is consistent with findings by Boriack-Sjodin et al. (2009), who demonstrated that methyltransferase enzymes influence histone activity and gene expression by adding methyl groups to DNA, proteins, and small molecule substrates [ 29 ]. The OTAbZIP gene may similarly modulate RNA metabolism and protein synthesis via methylation processes, influencing cellular response mechanisms to osmotic stress. Furthermore, previous studies by Zangari et al. (2014) and Lieu et al. (2017) highlighted the importance of pyruvate in cellular metabolism, particularly in glycolysis and energy production[ 30 , 31 ]. KEGG enrichment analysis revealed significant involvement of OTAbZIP in pathways such as DNA replication, fructose and mannose metabolism, pyruvate metabolism, and starch and sucrose metabolism. These findings suggest that the OTAbZIP gene plays a critical role in synthesizing hexoses like fructose and mannose, as well as pyruvate, which is essential for energy production and metabolic processes in A. westerdijkiae [ 32 ] Wang et al. reviewed showed that metal ion transport plays an important role in maintaining cellular metabolism and mitochondrial function [ 33 ]. Under 20 g/L NaCl, GO analysis identified significant changes in the biological processes of DNA replication, metal ion transport, nucleoside metabolism, and glycosyl complex metabolism. Notably, metal ion transport emerged as a critical pathway under this condition, likely due to the introduction of Na + ions, which affect cellular functions. The OTAbZIP gene appears to influence mitochondrial function and cellular metabolism by modulating the transport of Na + ions. Metal ion transport is closely linked to oxidative phosphorylation, enzyme activity, and cellular processes like apoptosis and cell proliferation, which are vital for stress response. In addition, the significance of differential gene expression in cellular components such as the cytoskeleton, ribosomal outer membrane, and endoplasmic reticulum membrane suggests that OTAbZIP affects these cellular structures under osmotic stress conditions. The impact of OTAbZIP on microtubule motility activity further implies that it may regulate mycelial growth and division, as well as processes related to metal ion transport and transmembrane transport activity[ 34 ]. This aligns with Winter et al. (2015), the role of arginine in fine-tuning developmental and defense mechanisms in response to stress [ 35 ]. KEGG enrichment analysis indicated that the OTAbZIP gene is involved in several metabolic pathways, including sugar metabolism, pyruvate metabolism, DNA replication, ribosome biogenesis, and arginine/proline metabolism. These processes are essential for nitrogen storage mobilization and cellular defense mechanisms, supporting the growth and stress tolerance of A. westerdijkiae . The chemical structure of small molecule metabolites often influences the regulatory network. NaCl and glucose tend to influence fungal growth and virulence production, as well as the activation of certain regulatory pathways [ 36 , 37 ]. At 100 g/L NaCl, significant changes were observed in pathways related to nucleoside metabolism, glycosyl compound metabolism, and small molecule metabolism, including nucleobases and carbohydrate derivatives. The differential expression of genes associated with membrane protein complexes and plasma membranes highlights the structural alterations that occur in response to osmotic stress. Furthermore, the down-regulation of oxidative phosphorylation and unsaturated fatty acid biosynthesis pathways suggests that OTAbZIP influences mitochondrial function and cellular energy production under extreme osmotic stress conditions. These findings suggest that the OTAbZIP gene plays a role in inhibiting the growth of A. westerdijkiae under high osmotic stress by regulating the synthesis of nucleobases, nucleosides, and nucleotides [ 38 ]. The significance of the differential genes for membrane protein complexes, plasma membranes, and cytoplasmic vesicles in terms of cellular composition indicates that the OTAbZIP gene affected the structure of membrane protein complexes, plasma membranes, and cytoplasmic vesicles under 100 g/L NaCl conditions. In terms of molecular function, the OTAbZIP gene may influence flavin adenine dinucleotide binding to regulate the redox state of mitochondria, which in turn influences RNA synthesis by endonuclease activity [ 39 ]. Nothling et al. reported that hydrolases are responsible for the catalyzed reactions of a wide range of lipids, sugars and proteins [ 40 ]. Hydrolytic enzymes play a crucial role in the breakdown of lipids, starch, and proteins, while monooxygenases are involved in redox reactions. The OTAbZIP gene may influence both hydrolytic enzyme activity and monooxygenase activity, potentially affecting protein synthesis and sugar metabolism in A. westerdijkiae . KEGG enrichment analysis suggests that the OTAbZIP gene is involved in acetaldehyde and dicarboxylate metabolism, oxidative phosphorylation, and the biosynthesis of unsaturated fatty acids under 100 g/L NaCl. These processes are linked to the glyoxylate cycle, which oxidatively breaks down fatty acids in A. westerdijkiae , providing essential raw materials for sugar synthesis [ 41 ]. The differential gene analysis of the HOG-MAPK pathway revealed that, under 0 g/L NaCl, the Hog1 gene and the phosphoglycerol dehydrogenase ( Gpd1 ) gene were significantly down-regulated in the ΔOTAbZIP mutant, indicating increased sensitivity to osmotic stress. Previous studies have shown that Hog1 is a key regulator of the osmotic stress response, and its down-regulation suggests impaired stress tolerance in the mutant strain [ 42 ].The Gpd1 gene, which mediates carbohydrate and lipid metabolism, is also down-regulated in the ΔOTAbZIP strain, highlighting its reduced ability to withstand osmotic stress [ 43 ]. Under 20 g/L NaCl, the expression of Cdc28 , a key protein in the cell cycle, was up-regulated in the mutant strain, suggesting that OTAbZIP may influence chromatin remodeling and DNA replication processes. In contrast, down-regulation of the Pbs2 and Hog1 genes in the Δ OTAbZIP strain suggests that the osmotic stress response pathway is compromised under moderate NaCl stress, although it still allows for adaptation and growth. The altered expression of Ctt1 , a catalase involved in oxidative stress protection, further supports the role of OTAbZIP in regulating cellular defense mechanisms under osmotic stress [ 44 – 46 ]. It has been reported that high osmolarity conditions increase tyrosine phosphorylation of Hog1 to induce peroxidase gene catalase expression to protect cell survival. In contrast, the down-regulated expression of the Ctt1 gene may be due to the increased osmotic pressure sensitivity of the mutant strain Δ OTAbZIP resulting in the strain itself adapting to the high osmotic pressure environment and thus by up-regulating the expression of the catalase gene ( Ctt1 ) [ 47 , 48 ]. Several Studies have shown that the Cdc28 -encoding gene is involved in several processes, including chromosome organisation, regulation of cell cycle processes and regulation of metabolic processes of nucleobase-containing compounds. Whereas up-regulation of Cdc28 encoding gene indicates that the OTAbZIP gene may be involved in the regulation of chromatin reorganization during DNA replication, down-regulation of the Cdc28 encoding gene for the mutant strain Δ OTAbZIP may lead to alteration of the meiotic process and DNA replication process, which affects the growth of mycelium [ 49 – 51 ]. Comparison of WT of A. westerdijkiae with the mutant strain Δ OTAbZIP under 100 g/L NaCl showed that the expression of the gene encoding mitogen-activated protein kinase ( Hog1 ) in the HOG-MAPK pathway was down-regulated suggesting that deletion of the OTAbZIP gene leads to an increased and relatively stable osmotic stress sensitivity in A. westerdijkiae. Protein-protein interaction (PPI) network analysis revealed a significant difference in protein connectivity under varying osmolarity conditions. Under 0 g/L NaCl, a relatively low number of interacting proteins was observed, whereas the number of interacting proteins was highest under 20 g/L NaCl, reflecting increased complexity in the stress response. At 100 g/L NaCl, the PPI network showed a decline in protein connectivity, suggesting a shift towards more specialized interactions under extreme osmotic conditions [ 52 , 53 ]. Finally, qRT-PCR analysis of the Hog1 gene and OTA biosynthesis genes ( otaA-D ) revealed that the expression of Hog1 was significantly higher in the Δ OTAbZIP mutant compared to the WT strain under all NaCl conditions. This indicates that the deletion of OTAbZIP enhances the osmotic stress response, potentially compensating for the absence of OTA production. The expression of the OTA biosynthesis genes in the Δ OTAbZIP strain was significantly lower than in the WT strain, confirming that the OTAbZIP gene is essential for OTA production under osmotic stress conditions [ 54 ]. It indicates that the OTAbZIP gene significantly affects the OTA biosynthesis genes ota ( A-D ), resulting in the inability of the mutant strain to produce OTA. Wang et al. reviewed the regulatory mechanisms of OTA and the role of biosynthetic genes[ 55 ]. Whereas otaD gene expression did not show a correlation with OTA production, the significant up-regulation of otaD gene expression under 100 g/L NaCl may be involved in the OTA chlorination step to maintain partial intracellular Cl − homeostasis. Our study highlights the critical role of the OTAbZIP gene in regulating key metabolic and stress response pathways in A. westerdijkiae , particularly under osmotic stress. The findings from this work provide new insights into the molecular mechanisms underlying OTA biosynthesis and stress tolerance, which may contribute to future strategies for controlling fungal growth and mycotoxin production in agricultural settings 4. Conclusion In this study, RNA-Seq was used to uncover transcriptional changes and key pathways in WT and ΔOTAbZIP mutant A. westerdijkiae fc-1 strains. The OTAbZIP gene was found to be involved in nucleoside metabolism, glycosyl complex metabolism, and DNA replication under 0 g/L NaCl conditions. At 20 g/L NaCl, OTAbZIP played a role in sugar and pyruvate metabolism, DNA replication, ribosomal function, and arginine and proline metabolism. Under 100 g/L NaCl conditions, OTAbZIP influenced glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, and the biosynthesis of unsaturated fatty acids, potentially affecting the growth and OTA production of A. westerdijkiae . Additionally, analysis of differential genes in the HOG pathway under varying osmotic pressures, along with qRT-PCR validation of OTA biosynthesis genes ( otaA - D ), revealed that deletion of the OTAbZIP gene significantly reduced OTA biosynthesis in the ΔOTAbZIP mutant strain. Moreover, the expression of Hog1 in the ΔOTAbZIP mutant was higher than in the WT strain, suggesting increased sensitivity to osmotic stress in the ΔOTAbZIP mutant. In conclusion, this study provides valuable insights into the role of OTAbZIP in the growth and mycotoxin production of A. westerdijkiae fc-1, laying a theoretical foundation for strategies aimed at reducing OTA contamination in food. 5 Materials and Methods 5.1 Strains and culture medium The WT OTA-producing strain A. westerdijkiae fc-1 (PRJNA264608), isolated from agricultural soil obtained from the Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing and the ΔOTAbZIP mutant A. westerdijkiae fc-1 strain [ 56 ] constructed by knocking out the OTAbZIP gene in our laboratory were inoculated into potato dextrose agar (PDA) medium. Both strains were incubated in the dark at 28°C for 7 days. Spores were collected using a sterile cotton swab, resuspended in sterile water and the spore concentration was adjusted to 10 7 spores /mL. Spore suspensions of both the strains were preserved at − 80°C with 15% glycerol. 5.2 Total RNA extraction, library construction and Illumina sequencing 5.2.1 Sample Preparation and RNA Extraction A 100 µL spore suspension of both the WT and ΔOTAbZIP mutant strains of A. westerdijkiae fc-1 was inoculated onto PDA medium and incubated in the dark at 28°C for 72 hours (h). After incubation, 100 mL of PDB medium with NaCl concentrations of 0, 20, and 100 g/L was prepared and sterilized. Each condition was replicated three times. Fresh mycelium from each strain was inoculated into the prepared PDB medium and incubated for 72 h in the dark at 28°C and 160 rpm. After incubation, fungal spores were collected by transferring the cultures to 100 mL centrifuge tubes and centrifuging at 10,000 rpm for 5 min. The supernatant was discarded, and the fungal spores were retained, filtered, and labeled for subsequent total RNA extraction. 5.2.2. Library Construction and Sequencing: The mRNA with a polyA tail was enriched using Oligo (dT) magnetic beads. The enriched mRNA was then fragmented using divalent cations in NEB Fragmentation Buffer following the NEB standard protocol[ 57 , 58 ]. First-strand cDNA was synthesized using M-MuLV reverse transcriptase, with fragmented mRNA as the template and random oligonucleotides as primers. The RNA strand was then degraded with RNaseH, and the second strand of cDNA was synthesized using dNTPs and DNA polymerase I. The resulting double-stranded cDNA was purified, end-repaired, A-tailed, and ligated to sequencing adapters. Subsequently, cDNA fragments around 250–300 bp were selected using AMPure XP beads, followed by PCR amplification. The PCR products were purified again using AMPure XP beads to obtain the final libraries [ 59 ]. The kit used for library construction was NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. The libraries were quantified preliminarily using a Qubit 2.0 Fluorometer and diluted to a concentration of 1.5 ng/µL. The insert size was assessed using an Agilent 2100 bioanalyzer, and once the size met expectations, the effective concentration of the library was determined by qRT-PCR (ensuring it was above 2 nM) to confirm library quality. 5.3 GO functional annotation of DEGs and KEGG pathway differential gene analysis GO functional enrichment analysis of differentially expressed genes (DEGs) was performed using the cluster Profiler software (3.8.1ver.) [ 57 ]. This enrichment analysis is based on the hypergeometric distribution principle. The differential gene set consists of genes identified as differentially expressed and annotated in the GO database. In contrast, the background gene set includes all genes analyzed for differential significance and annotated in the GO database. A padj value of less than 0.05 was used as the threshold for significant enrichment. The 30 most significant GO Terms were selected for visualization in histograms; if fewer than 30 were identified, all were displayed. Histograms were categorized according to the three major GO categories: biological processes, cellular components, and molecular functions, along with the classification of up-and down-regulated differential genes. KEGG pathway enrichment analysis was conducted on the differentially expressed genes of the A. westerdijkiae fc-1 strain and ΔOTAbZIP mutant A. westerdijkiae strain under three conditions (0, 20, 100 g/L) of NaCl concertation. The analysis was performed using Cluster Profiler software (3.8.1ver.), with a significant threshold of padj less than 0.05. From the KEGG pathway enrichment results, the top 30 significant pathways were selected to create a scatterplot representation. 5.4 Differential Gene Protein Interaction Network Analysis The interactions in the STRING protein interaction database were applied for the analysis of differential gene protein interaction networks. For species included in the database, the interactions of the target gene set were directly extracted from the database to construct the network; for species not included in the database, we firstly applied blastx comparison of the sequences in the target gene set to the protein sequences of the reference species included in the STRING database and constructed the interactions network by using the protein interactions of this reference species on the comparison. Visual editing was performed using Cytoscape software. 5.5 Validation of RNA-Seq analysis by RT-qPCR The WT and Δ OTAbZIP mutant of A. westerdijkiae fc-1 strains were inoculated into PDB medium supplemented with NaCl at concentrations of 0, 20, and 100 g/L. Cultures were incubated at 28°C and 180 rpm for 3 days post-inoculation (dpi). Equal amounts of mycelium were collected from each culture and homogenized in 1 mL Trizol reagent. Samples were incubated at room temperature for 5 min to ensure complete lysis. The homogenates were centrifuged at 12,000 rpm and 4°C for 5 min, and the supernatants were transferred to new 1.5 mL centrifuge tubes. Chloroform (1:5 v/v relative to Trizol) was added, followed by gentle inversion for 30 seconds. Samples were left at room temperature for 5 min to separate phases without agitation to minimize genomic DNA shearing. The upper aqueous phase was carefully transferred to a new tube, mixed with isopropanol (0.5–1× the volume of Trizol), and incubated at room temperature for 10 min. RNA was pelleted by centrifugation at 12,000 rpm and 4°C for 10 min. The RNA pellet was washed with 75% ethanol (same volume as Trizol), centrifuged at 7,500 rpm and 4°C for 5 minutes, and air-dried or vacuum-dried for 5–10 minutes. The RNA was resuspended in an appropriate volume of DEPC-treated water. RNA integrity was confirmed by electrophoresis, and concentrations were measured using a Quawell Q3000 ultra-micro UV spectrophotometer (Table 3 ). To analyze gene expression, 1 µg of RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Life Technologies, Milan, Italy). Expression levels of OTA synthesis genes ( otaA , otaB , otaC , and otaD ) and Hog1 were quantified using a Real-Time PCR system with primers listed in Table 2 . Amplification conditions were initial denaturation at 95°C for 3 minutes, followed by 35 cycles of 95°C for 10 seconds, 60°C for 45 seconds, and a final extension at 72°C for 7 minutes. Relative expression levels were calculated using the 2-ΔΔCT method with CFX Manager software (Bio-Rad Laboratories) [ 60 ]. Each treatment was performed in three biological replicates. Table 3 RNA concentrations after extraction measured by Quawell-Q3000 ultra-micro UV spectrophotometer Sample ID Sample Type 260/280 Concentration WT_0 g/L NaCl RNA 1.54 56.80 Δ OTAbZIP _0 g/L NaCl RNA 1.61 325.70 WT_20 g/L NaCl RNA 1.61 120.10 Δ OTAbZIP _20 g/L NaCl RNA 1.70 368.00 WT_100 g/L NaCl RNA 1.82 397.40 Δ OTAbZIP _20 g/L NaCl RNA 1.82 1135.80 Declarations Conflict of interest: All authors have no conflict of interest and agree to the published version of the manuscript. Funding: This research was funded by National Key Research and Development Program of China (2022YFE0139500). 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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-5792788","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":400774616,"identity":"8c70147b-87b4-44ba-98ad-20012db2fd54","order_by":0,"name":"Yanling Ma","email":"","orcid":"","institution":"Foshan University","correspondingAuthor":false,"prefix":"","firstName":"Yanling","middleName":"","lastName":"Ma","suffix":""},{"id":400774617,"identity":"19d933d0-4d96-4d56-8956-ec40b0836b14","order_by":1,"name":"Muyuan Zhuang","email":"","orcid":"","institution":"Foshan University","correspondingAuthor":false,"prefix":"","firstName":"Muyuan","middleName":"","lastName":"Zhuang","suffix":""},{"id":400774618,"identity":"773f62e0-768a-457a-b36b-482bb9b5a650","order_by":2,"name":"Tanvir Ahmad","email":"","orcid":"","institution":"Foshan University","correspondingAuthor":false,"prefix":"","firstName":"Tanvir","middleName":"","lastName":"Ahmad","suffix":""},{"id":400774619,"identity":"564344f4-7930-4ee5-966d-2c5c38489c77","order_by":3,"name":"Yuhong Yan","email":"","orcid":"","institution":"Foshan University","correspondingAuthor":false,"prefix":"","firstName":"Yuhong","middleName":"","lastName":"Yan","suffix":""},{"id":400774620,"identity":"3828a1b4-94c0-4e94-acb5-90cbe5b50816","order_by":4,"name":"Weitian Yuan","email":"","orcid":"","institution":"Foshan University","correspondingAuthor":false,"prefix":"","firstName":"Weitian","middleName":"","lastName":"Yuan","suffix":""},{"id":400774621,"identity":"596059be-5d13-4500-860a-aa1d0136e26d","order_by":5,"name":"Mingxuan Li","email":"","orcid":"","institution":"Foshan University","correspondingAuthor":false,"prefix":"","firstName":"Mingxuan","middleName":"","lastName":"Li","suffix":""},{"id":400774622,"identity":"c9c0236d-00ad-42e0-8c89-a1806ab2019c","order_by":6,"name":"Guangyou Tan","email":"","orcid":"","institution":"Foshan University","correspondingAuthor":false,"prefix":"","firstName":"Guangyou","middleName":"","lastName":"Tan","suffix":""},{"id":400774623,"identity":"d9bf1003-2909-4a1a-9ec0-b1a5d51f9c11","order_by":7,"name":"Yingyao Deng","email":"","orcid":"","institution":"Foshan University","correspondingAuthor":false,"prefix":"","firstName":"Yingyao","middleName":"","lastName":"Deng","suffix":""},{"id":400774624,"identity":"67fd87e0-d49b-482f-9504-9e9608d29526","order_by":8,"name":"Yang Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYBACPmYILcfeAKaZCWthg6ox5jlAtBYondhDvBZ2HjOJnztq03vY259JMFRYJzawnz1AwGE8ZpK9Z47n9vCcMZNgOJOe2MCTl0BQiwRv27Hc/RI5bBKMbYcTGyR4DAjb8rftWDqPRPozCcZ/RGqR5m2rSeCRSDCTYGwgSgtbsbVs2wFDoF+MLRKOpRu38eTg18LPf3jjzbdtdfI87O0Pb3yosZbtZz+DXwsQsEgwMByGMBMYEDGFDzB/YGCoI0LdKBgFo2AUjFgAAPULOe3545NqAAAAAElFTkSuQmCC","orcid":"","institution":"Foshan University","correspondingAuthor":true,"prefix":"","firstName":"Yang","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-01-09 03:23:06","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5792788/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5792788/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":73695977,"identity":"2644efd7-fcf2-45fd-8e5b-7d2b43253173","added_by":"auto","created_at":"2025-01-13 16:19:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147979,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of differentially expressed genes (Set 1-9) in WT and mutant Δ\u003cem\u003eOTAbZIP\u003c/em\u003e under (A) 0, (B) 20 and (C) 100 g/L NaCl. 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The redder the color the higher the expression, and the greener the lower the expression.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5792788/v1/7b0506a82d73a70eb8847bf0.png"},{"id":73695983,"identity":"818c5ac6-2f80-4316-896b-d86566d786fc","added_by":"auto","created_at":"2025-01-13 16:19:12","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":245368,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential gene expression comparisons of\u003cem\u003eA. westerdijkiae\u003c/em\u003e WT strain and Δ\u003cem\u003eOTAbZIP \u003c/em\u003emutant strain at (A) 0, (B) 20, and (C) 100 g/L NaCl concentration, 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5","display":"","copyAsset":false,"role":"figure","size":404940,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG-enriched pathways of differential genes in \u003cem\u003eA. westerdijkiae \u003c/em\u003eWT\u003cem\u003e \u003c/em\u003estrain and Δ\u003cem\u003eOTAbZIP\u003c/em\u003e mutant strain at (A) 0 (B) 20 and (C) 100 g/L NaCl concentrations.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-5792788/v1/1c2bffeeb472ea9542649d08.png"},{"id":73695985,"identity":"7a2877f4-8de4-43c2-8a20-8f29b567e4a6","added_by":"auto","created_at":"2025-01-13 16:19:12","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":421639,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG-enriched pathways of differential genes in \u003cem\u003eA. westerdijkiae\u003c/em\u003e WT strain and Δ\u003cem\u003eOTAbZIP\u003c/em\u003emutant strain at (A) 0, (B) 20, and (C) 100 g/L NaCl concentrations.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-5792788/v1/c0bf1cfd9d653347e8566b28.png"},{"id":73697066,"identity":"e28b2f77-36c8-47ba-ae84-498e1f3270bc","added_by":"auto","created_at":"2025-01-13 16:27:12","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":765742,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential Gene Protein Interaction Network under (A) 0 (B) 20 and (C) 100 g/L NaCl.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-5792788/v1/e62e1b51117d9c4f1f86f6e8.png"},{"id":73697068,"identity":"7a720ece-6596-4bb8-97df-eb1089707d79","added_by":"auto","created_at":"2025-01-13 16:27:12","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":318791,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFig.7.\u003c/strong\u003e The expression of (A) \u003cem\u003eHog1\u003c/em\u003e and OTA biosynthesis genes (B) \u003cem\u003eotaA\u003c/em\u003e, (C) \u003cem\u003eotaB\u003c/em\u003e, (D) \u003cem\u003eotaC\u003c/em\u003e and (E) \u003cem\u003eotaD\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-5792788/v1/e192f69632bcfa31c2d00dd3.png"},{"id":76401448,"identity":"45e86a4b-0a55-4539-88ab-fa0dd12d4bec","added_by":"auto","created_at":"2025-02-16 16:08:34","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4152728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5792788/v1/836db0df-73db-4d24-ac4a-3549502c5a5c.pdf"},{"id":73698671,"identity":"87c3ff9f-9516-4b50-b550-b5c2a0f0008f","added_by":"auto","created_at":"2025-01-13 16:43:12","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":316460,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGraphical Abstract\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"GA.png","url":"https://assets-eu.researchsquare.com/files/rs-5792788/v1/3b835ef7238138486c33be16.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Role of the OTAbZIP Gene in Regulating Growth and OTA Production in Aspergillus westerdijkiae fc-1 Under Osmotic Stress","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOchratoxin A (OTA) is one of the most prevalent mycotoxins in food and feed. It is a low molecular weight secondary metabolite produced by certain species of \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003ePenicillium\u003c/em\u003e [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. OTA commonly contaminates cereals, pulses, dried fruits, grapes, tea, and animal feeds. Human and animal exposure to OTA has been associated with acute and chronic toxicity, including carcinogenicity and renal dysfunction [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. OTA contamination primarily occurs through the consumption of naturally contaminated food and feed. In 2016, the International Agency for Research on Cancer (IARC) classified OTA as a Group 2B carcinogen [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e is one of the most important OTA-producing species of \u003cem\u003eAspergillus\u003c/em\u003e and a significant contaminant of food products [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recent studies have shown that \u003cem\u003eAwAreA\u003c/em\u003e, a nitrogen-responsive transcriptional regulator in \u003cem\u003eA. westerdijkiae\u003c/em\u003e, plays a crucial role in regulating nitrogen sources. It significantly impacts the strain's growth and conidial formation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Under high osmotic stress, \u003cem\u003eΔAwSakA\u003c/em\u003e mutants of \u003cem\u003eA. westerdijkiae\u003c/em\u003e exhibited reduced mycelial growth and decreased expression of the biosynthetic genes \u003cem\u003eotaA\u003c/em\u003e, \u003cem\u003eotaB\u003c/em\u003e, and \u003cem\u003eotaD\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The absence of the transcription factor \u003cem\u003eAwHog1\u003c/em\u003e in \u003cem\u003eA. westerdijkiae\u003c/em\u003e also resulted in reduced mycelial growth and reduced OTA production [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eAspergillus\u003c/em\u003e species are constantly influenced by various environmental factors, with osmotic pressure being one of the most critical. This factor plays a pivotal role in regulating their mycelial growth and mycotoxin production [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The mitogen-activated protein kinase (MAPK) signaling pathway is widespread and highly conserved in eukaryotic cells. It regulates transcriptional activity and biochemical responses by phosphorylating downstream transcription factors. Additionally, it modulates various enzymes, enabling the cell to adapt to changes in the external environment [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. The \u003cem\u003eHog1\u003c/em\u003e-MAPK signaling pathway can be activated by extracellular high osmotic stress and plays a crucial role in protecting cells in high osmotic pressure environments [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The \u003cem\u003eOTAbZIP\u003c/em\u003e gene encodes a basic leucine zipper (\u003cem\u003ebZIP\u003c/em\u003e) transcription factor, which is one of the most widely distributed types of transcription factors in eukaryotes. The \u003cem\u003ebZIP\u003c/em\u003e transcription factors regulate gene expression by binding to cis-acting elements within promoter sequences upstream of structural genes, playing a critical role in activating or repressing transcription [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. The \u003cem\u003eOTAbZIP\u003c/em\u003e gene also controls the expression of the OTA biosynthesis genes [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Osmotic pressure signaling is essential for maintaining cellular homeostasis and development in fungi. The \u003cem\u003ebZIP\u003c/em\u003e transcription factors are key players in the regulatory networks of this signaling [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. \u003cem\u003eA. westerdijkiae Sarcolamban B\u003c/em\u003e (\u003cem\u003eAwSclB\u003c/em\u003e) gene may have a negative regulatory effect on OTA synthesis through its interaction with \u003cem\u003eOTAbZIP\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The \u003cem\u003eatfA\u003c/em\u003e gene of \u003cem\u003ePenicillium marneffei\u003c/em\u003e, which encodes a \u003cem\u003ebZIP\u003c/em\u003e-type transcription factor, was characterized. The \u003cem\u003eatfA\u003c/em\u003e mutant exhibited increased sensitivity to sodium dodecyl sulfate and tert-butyl hydroperoxide compared to wild-type strains. However, it showed no sensitivity to sodium chloride, sorbitol, ultraviolet light and heat stress.[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The \u003cem\u003ebZIP\u003c/em\u003e transcription factor \u003cem\u003eatf1\u003c/em\u003e in wood fungi plays a key role in blue light response and MAPK \u003cem\u003eHOG1\u003c/em\u003e downstream signaling [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. However, the impact of the \u003cem\u003eOTAbZIP\u003c/em\u003e gene on differential gene expression within the mycotoxin-producing metabolic pathway and the HOG pathway of \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 strains under varying osmolarity conditions remains unclear. To explore this, a mutant strain of Δ\u003cem\u003eOTAbZIP A. westerdijkiae\u003c/em\u003e was constructed to investigate how the \u003cem\u003eOTAbZIP\u003c/em\u003e gene influences metabolic pathways and gene expression related to OTA production. The recent advancements in sequencing technology, RNA-Seq has emerged as a cost-effective tool that greatly enhances bioinformatics analysis [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe objectives of this study were to investigate the role of the environmental regulator \u003cem\u003eOTAbZIP\u003c/em\u003e gene in \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 under different osmotic pressure conditions using transcriptome sequencing (RNA-Seq). Specifically, we aimed to determine how the \u003cem\u003eOTAbZIP\u003c/em\u003e gene affects metabolic pathways related to mycelium growth and OTA production, with a focus on the HOG pathway and differentially expressed genes. Additionally, we analyzed the expression of \u003cem\u003eHog1\u003c/em\u003e and OTA biosynthesis genes, key components of the HOG pathway, through RT-PCR to verify the impact of \u003cem\u003eOTAbZIP\u003c/em\u003e on these crucial genes. The results will help to refine the role of the \u003cem\u003eOTAbZIP\u003c/em\u003e gene in regulating metabolic pathways related to the growth and virulence production of \u003cem\u003eA. westerdijkiae\u003c/em\u003e strains under different osmotic pressure conditions.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Analysis of RNA sequencing results\u003c/h2\u003e \u003cp\u003eThe clean reads were obtained from 18 samples after filtering out low-quality sequences (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All bases met the Q30 criteria, exceeding 91%, and the GC content ranged between 35% and 65%. Finally, a total of 79,305,730 and 302 genes were identified, with an average length of 643.2 bp. These results indicated high sequencing quality, suitable for further analysis.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSummary of RNA sequencing metrics for WT and \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant strains under different NaCl concentrations*.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eClean reads\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eError rate (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eQ\u0026thinsp;\u0026gt;\u0026thinsp;30 (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGC content (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT_0 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43632895\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔ\u003cem\u003eOTAbZIP\u003c/em\u003e_0 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e43858039\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e91.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e50.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT_20 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e41636893\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.06\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e49.32\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔ\u003cem\u003eOTAbZIP\u003c/em\u003e_20 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42754401\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e47.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT_100 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e44531399\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e51.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔ\u003cem\u003eOTAbZIP\u003c/em\u003e_100 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e42993410\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e92.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e45.79\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003e*Q \u0026gt; 30 is the percentage of bases with a mass value greater than 30 in the total bases with an error rate \u0026lt; 0.1%; GC is the percentage of G and C bases in the total bases in the number of reads after filtering\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Differential gene expression\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. Clustering Analysis\u003c/h2\u003e \u003cp\u003eDifferential genes were compiled from all comparison groups, allowing for cluster analysis of expression patterns across six sample sets. A heatmap was generated to illustrate gene clustering, where color intensity reflects normalized expression values (typically between \u0026minus;\u0026thinsp;2 and 2). The heatmap enables horizontal comparisons of gene expression across different samples.\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Volcano Plot Analysis\u003c/h2\u003e \u003cp\u003eVolcano plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) visualize the distribution of differential gene expression across various comparisons. The horizontal axis represents the log\u003csup\u003e2\u003c/sup\u003e fold change in gene expression between treated and control groups while the vertical axis indicates the significance of these differences (-log10 adjusted \u003cem\u003ep-\u003c/em\u003evalue). Up-regulated genes are marked with red dots and down-regulated genes with green dots. In the comparison of \u003cem\u003eA. westerdijkiae\u003c/em\u003e WT and mutant Δ\u003cem\u003eOTAbZIP\u003c/em\u003e at 0 g/L NaCl, a total of 13,294 differentially expressed genes (DEGs) were identified, including 968 up-regulated and 1,254 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). While under 20 g/L NaCl, a total 13,200 DEGs were observed, with 1,575 up-regulated and 2,033 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). At 100 g/L NaCl, a total of 12,585 DEGs were found, comprising 208 up-regulated and 316 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Differential gene GO function analysis\u003c/h2\u003e \u003cp\u003eGene Ontology (GO) analysis categorizes gene functions into biological processes, cellular components, and molecular functions, with results displayed in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e (A, B, C). The x-axis indicates the significance level of GO term enrichment, with higher values denoting greater significance. The y-axis lists the GO terms and color coding differentiates between biological processes (BP), cellular components (CC), and molecular functions (MF), with a significance threshold set at padj\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003eIn the 0 g/L NaCl comparison, significant biological processes included nucleoside metabolism, glycosyl complex metabolism, and DNA replication, with no notable changes in cellular composition. Molecular functions such as oxidoreductase activity and RNA endonuclease activity also showed significance (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). At 20 g/L NaCl, significant processes included DNA replication and metal ion transport, alongside notable cellular components like the cytoskeleton and endoplasmic reticulum membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). At 100 g/L NaCl, significant processes related to nucleoside metabolism and carbohydrate derivatives emerged, with notable changes in membrane protein complexes and other cellular components (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eDifferential genes in biological processes such as nucleoside metabolic processes, glycosyl complex metabolic processes and DNA replication were significant in \u003cem\u003eA. westerdijkiae\u003c/em\u003e WT compared with the mutant Δ\u003cem\u003eOTAbZIP\u003c/em\u003e strain under 0 g/L NaCl. No significant changes in physiological function in terms of cellular composition were produced. Oxidoreductase activity, ribonucleic acid endonuclease activity, methyltransferase activity, and transmembrane transporter protein activity were shown to have significant plots for genes differing in molecular function.\u003c/p\u003e \u003cp\u003eDifferential genes in biological processes such as DNA replication, metal ion transport, transporter nucleoside metabolism processes, and glycosyl complex metabolism processes were significant in \u003cem\u003eA.westerdijkiae\u003c/em\u003e wild-type WT compared with the mutant strain Δ\u003cem\u003eOTAbZIP\u003c/em\u003e at 20 g/L NaCl. Differential genes were significant in cellular components such as cytoskeleton, ribosomal outer membrane, endoplasmic reticulum membrane. Differential genes were significant in molecular functions such as metal ions, transmembrane transporter activity, microtubule motility activity, and antioxidant activity\u003c/p\u003e \u003cp\u003eDifferential genes in biological processes such as nucleoside metabolism processes, glycosyl compound metabolism processes, small molecule metabolism processes containing nucleobases, and carbohydrate derivative metabolism processes were significant in \u003cem\u003eA.westerdijkiae\u003c/em\u003e wild-type WT compared with the mutant strain Δ\u003cem\u003eOTAbZIP\u003c/em\u003e at 100 g/L NaCl. Differential genes in membrane protein complexes, plasma membranes, cytoplasmic vesicles, and other cellular components were significant. Differential genes in molecular functions such as flavin adenine dinucleotide binding, endonuclease activity, monooxygenase activity, and hydrolase activity have significant plots.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Functional analysis of the differential gene via KEGG\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e illustrates the KEGG enrichment results, highlighting the 20 most significant pathways. The x-axis reflects the ratio of annotated differential genes to the total number of DEGs, while the y-axis lists the KEGG pathways. The size of the dots indicates the number of genes associated with each pathway, and colors range from red to purple to represent enrichment significance.\u003c/p\u003e \u003cp\u003eKey KEGG pathways enriched at 0 g/L NaCl included DNA replication, fructose and mannose metabolism, steroid biosynthesis, pyruvate metabolism, and starch and sucrose metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). At 20 g/L NaCl, significant pathways encompass sugar metabolism, ribosomal processes, and arginine and proline metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). At 100 g/L NaCl, differential genes were enriched in pathways related to acetate and dicarboxylate metabolism, oxidative phosphorylation, and unsaturated fatty acid biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC).\u003c/p\u003e\u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 HOG-MAPK pathway differential gene analysis\u003c/h2\u003e \u003cp\u003eThe results of the KEGG pathway analysis highlighting the HOG-MAPK pathway are illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. In this figure, KEGG nodes representing up-regulated genes are denoted in red, while those representing down-regulated genes are marked in green. The shading of the colors corresponds to the significance level of the gene expressions. The mitogen-activated protein kinase (MAPK) signaling pathway is a highly conserved and ubiquitous system in eukaryotic cells, playing a crucial role in cellular signal transduction. Specifically, the Hog1 MAPK pathway is activated by external stimuli, such as high osmotic stress, and is vital for cell survival in hyperosmotic conditions [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Previous studies have demonstrated that the NaCl-induced production of OTA is associated with the phosphorylation status of the HOG MAP kinase [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Under 0g/L NaCl, the comparison between the wild-type (WT) and the mutant ΔOTAbZIP strain revealed a down-regulation of the mitogen-activated protein kinase-encoding gene \u003cem\u003eHog1\u003c/em\u003e, alongside a significant down-regulation of the glycerol synthetase-encoding gene \u003cem\u003eGpd1\u003c/em\u003e within the HOG-MAPK pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). while under 20 g/L NaCl, the analysis showed an up-regulation of the HOG-MAPK pathway, particularly involving the cell cycle protein-dependent serine \u003cem\u003eCdc28\u003c/em\u003e. Conversely, there was down-regulation observed in the genes encoding \u003cem\u003eHog1\u003c/em\u003e, the protein kinase \u003cem\u003ePbs2\u003c/em\u003e, and the catalase gene \u003cem\u003eCtt1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB). At under 100 g/L NaCl, the comparison indicated down-regulation of the \u003cem\u003eHog1\u003c/em\u003e gene within the HOG-MAPK pathway, consistent with the previous findings under varying NaCl concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Differential Gene Protein Interaction Network\u003c/h2\u003e \u003cp\u003eProtein-protein interactions (PPIs) played important roles in the molecular recognition of pathogens. Figure\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003e represented the PPIs network views of the WT strain and mutant \u003cem\u003eΔOTAbZIP\u003c/em\u003e strain under 0, 20, and 100 g/L NaCl conditions, respectively. Each node in the interworking network graph represents a protein, the size of the node is proportional to the degree of this node, and the color gradient of the node from yellow to red corresponds to the value of the aggregation factor from low to high. By mapping the differential gene-protein interactions network, we found that the candidate gene under the 0 g/L NaCl condition was TU AoFC_00282 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and under the 20 g/L NaCl condition, it was TU AoFC_06641 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) while under the 100 g/L NaCl condition, it was TU AoFC_09006 and TU AoFC_09007 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e7\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7 OTA biosynthetic genes and \u003cem\u003eHog1\u003c/em\u003e gene expression under different osmolarity conditions\u003c/h2\u003e \u003cp\u003eThe expression levels of OTA biosynthesis genes (\u003cem\u003eotaA-D\u003c/em\u003e) and the \u003cem\u003eHog1\u003c/em\u003e gene were analyzed using quantitative reverse transcription PCR (qRT-PCR), as presented in Fig.\u0026nbsp;8. At 0 g/L NaCl, comparison of the wild-type (WT) strain of \u003cem\u003eA.westerdijkiae\u003c/em\u003e fc-1 with the mutant strain Δ\u003cem\u003eOTAbZIP\u003c/em\u003e revealed that the transcriptional expression level of the \u003cem\u003eHog1\u003c/em\u003e gene in the mutant strain increased by 26.02%. In contrast, the expression levels of the OTA biosynthesis genes showed significant decreases: \u003cem\u003eotaA\u003c/em\u003e decreased by 3.19-fold, \u003cem\u003eotaB\u003c/em\u003e by 2.64-fold, and \u003cem\u003eotaC\u003c/em\u003e by 5.51-fold, while the expression of \u003cem\u003eotaD\u003c/em\u003e in the Δ\u003cem\u003eOTAbZIP\u003c/em\u003e mutant strain increased by 23.65% compared to the WT strain. At 20 g/L NaCl, the comparison again demonstrated an increase in the transcriptional expression of the \u003cem\u003eHog1\u003c/em\u003e gene in the mutant Δ\u003cem\u003eOTAbZIP\u003c/em\u003e, approximately 1.12-fold compared to the WT. However, the OTA biosynthesis genes experienced dramatic decreases: \u003cem\u003eotaA\u003c/em\u003e decreased by 8,360-fold, \u003cem\u003eotaB\u003c/em\u003e by 24.82-fold, \u003cem\u003eotaC\u003c/em\u003e by 160.42-fold, and \u003cem\u003eotaD\u003c/em\u003e by 40.58-fold. At 100 g/L NaCl, the expression level of the Hog1 gene in the Δ\u003cem\u003eOTAbZIP\u003c/em\u003e mutant strain increased by 40.77% compared to the WT, while the OTA biosynthesis genes again showed substantial down-regulation, with \u003cem\u003eotaA\u003c/em\u003e decreasing by 5.34-fold, \u003cem\u003eotaB\u003c/em\u003e by 3.77-fold, \u003cem\u003eotaC\u003c/em\u003e by 12.12-fold, and \u003cem\u003eotaD\u003c/em\u003e by 4.02-fold. Overall, these results indicate that increasing NaCl concentrations significantly affect the transcriptional expression of both the \u003cem\u003eHog1\u003c/em\u003e gene and the OTA biosynthesis genes, with distinct patterns observed in the wild-type versus mutant strains.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eThis study used RNA-Seq technology to investigate the differentially expressed genes in WT and Δ\u003cem\u003eOTAbZIP\u003c/em\u003e mutant \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 strains. These strains were examined under different NaCl concentration conditions. KEGG pathway analysis was also performed to gain a more comprehensive and accurate understanding of how the \u003cem\u003eOTAbZIP\u003c/em\u003e gene regulates OTA production under varying osmotic stress conditions [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Notably, the DEGs in both WT and \u003cem\u003eΔOTAbZIP\u003c/em\u003e strains varied significantly under the different NaCl concentrations, indicating a substantial influence of osmotic stress on gene expression [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. M\u0026ouml;hlmann et al. (2015) demonstrated that nucleotides participate in various cellular processes, including nucleoside monophosphate synthesis and the modification of nucleic acids, which aligns with the transcriptomic analysis [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. At 0 g/L NaCl, GO functional analysis revealed significant changes in the biological processes of nucleoside metabolism, glycosyl complex metabolism, and DNA replication in both WT and \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant strains. These results suggest that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene influences cellular reactions by modulating glycosyl complex metabolism, which impacts purine and pyrimidine nucleotide metabolism, including DNA replication. Maintaining nucleotide metabolism balance appears to be essential for the proper development of \u003cem\u003eA. westerdijkiae\u003c/em\u003e. In line with this, a recent study by Sell\u0026eacute;s Vidal et al. (2021) emphasized the role of oxidoreductases in central metabolic pathways [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Interestingly, the lack of significant changes in bio-composition under varying NaCl conditions suggests that the deletion of \u003cem\u003eOTAbZIP\u003c/em\u003e did not drastically affect the overall physiological functions of \u003cem\u003eA. westerdijkiae\u003c/em\u003e. However, in terms of molecular function, the differential expression of genes involved in oxidoreductase activity, ribonuclease activity, methyltransferase activity, and transmembrane transporter protein activity indicates that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene likely regulates several key metabolic pathways. This is consistent with findings by Boriack-Sjodin et al. (2009), who demonstrated that methyltransferase enzymes influence histone activity and gene expression by adding methyl groups to DNA, proteins, and small molecule substrates [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. The \u003cem\u003eOTAbZIP\u003c/em\u003e gene may similarly modulate RNA metabolism and protein synthesis via methylation processes, influencing cellular response mechanisms to osmotic stress. Furthermore, previous studies by Zangari et al. (2014) and Lieu et al. (2017) highlighted the importance of pyruvate in cellular metabolism, particularly in glycolysis and energy production[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. KEGG enrichment analysis revealed significant involvement of \u003cem\u003eOTAbZIP\u003c/em\u003e in pathways such as DNA replication, fructose and mannose metabolism, pyruvate metabolism, and starch and sucrose metabolism. These findings suggest that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene plays a critical role in synthesizing hexoses like fructose and mannose, as well as pyruvate, which is essential for energy production and metabolic processes in \u003cem\u003eA. westerdijkiae\u003c/em\u003e [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] Wang et al. reviewed showed that metal ion transport plays an important role in maintaining cellular metabolism and mitochondrial function [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Under 20 g/L NaCl, GO analysis identified significant changes in the biological processes of DNA replication, metal ion transport, nucleoside metabolism, and glycosyl complex metabolism. Notably, metal ion transport emerged as a critical pathway under this condition, likely due to the introduction of Na\u0026thinsp;+\u0026thinsp;ions, which affect cellular functions. The \u003cem\u003eOTAbZIP\u003c/em\u003e gene appears to influence mitochondrial function and cellular metabolism by modulating the transport of Na\u0026thinsp;+\u0026thinsp;ions. Metal ion transport is closely linked to oxidative phosphorylation, enzyme activity, and cellular processes like apoptosis and cell proliferation, which are vital for stress response. In addition, the significance of differential gene expression in cellular components such as the cytoskeleton, ribosomal outer membrane, and endoplasmic reticulum membrane suggests that \u003cem\u003eOTAbZIP\u003c/em\u003e affects these cellular structures under osmotic stress conditions.\u003c/p\u003e \u003cp\u003eThe impact of \u003cem\u003eOTAbZIP\u003c/em\u003e on microtubule motility activity further implies that it may regulate mycelial growth and division, as well as processes related to metal ion transport and transmembrane transport activity[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. This aligns with Winter et al. (2015), the role of arginine in fine-tuning developmental and defense mechanisms in response to stress [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. KEGG enrichment analysis indicated that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene is involved in several metabolic pathways, including sugar metabolism, pyruvate metabolism, DNA replication, ribosome biogenesis, and arginine/proline metabolism. These processes are essential for nitrogen storage mobilization and cellular defense mechanisms, supporting the growth and stress tolerance of \u003cem\u003eA. westerdijkiae\u003c/em\u003e. The chemical structure of small molecule metabolites often influences the regulatory network. NaCl and glucose tend to influence fungal growth and virulence production, as well as the activation of certain regulatory pathways [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAt 100 g/L NaCl, significant changes were observed in pathways related to nucleoside metabolism, glycosyl compound metabolism, and small molecule metabolism, including nucleobases and carbohydrate derivatives. The differential expression of genes associated with membrane protein complexes and plasma membranes highlights the structural alterations that occur in response to osmotic stress. Furthermore, the down-regulation of oxidative phosphorylation and unsaturated fatty acid biosynthesis pathways suggests that \u003cem\u003eOTAbZIP\u003c/em\u003e influences mitochondrial function and cellular energy production under extreme osmotic stress conditions. These findings suggest that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene plays a role in inhibiting the growth of \u003cem\u003eA. westerdijkiae\u003c/em\u003e under high osmotic stress by regulating the synthesis of nucleobases, nucleosides, and nucleotides [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The significance of the differential genes for membrane protein complexes, plasma membranes, and cytoplasmic vesicles in terms of cellular composition indicates that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene affected the structure of membrane protein complexes, plasma membranes, and cytoplasmic vesicles under 100 g/L NaCl conditions. In terms of molecular function, the \u003cem\u003eOTAbZIP\u003c/em\u003e gene may influence flavin adenine dinucleotide binding to regulate the redox state of mitochondria, which in turn influences RNA synthesis by endonuclease activity [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Nothling et al. reported that hydrolases are responsible for the catalyzed reactions of a wide range of lipids, sugars and proteins [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Hydrolytic enzymes play a crucial role in the breakdown of lipids, starch, and proteins, while monooxygenases are involved in redox reactions. The \u003cem\u003eOTAbZIP\u003c/em\u003e gene may influence both hydrolytic enzyme activity and monooxygenase activity, potentially affecting protein synthesis and sugar metabolism in \u003cem\u003eA. westerdijkiae\u003c/em\u003e. KEGG enrichment analysis suggests that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene is involved in acetaldehyde and dicarboxylate metabolism, oxidative phosphorylation, and the biosynthesis of unsaturated fatty acids under 100 g/L NaCl. These processes are linked to the glyoxylate cycle, which oxidatively breaks down fatty acids in A. \u003cem\u003ewesterdijkiae\u003c/em\u003e, providing essential raw materials for sugar synthesis [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe differential gene analysis of the HOG-MAPK pathway revealed that, under 0 g/L NaCl, the \u003cem\u003eHog1\u003c/em\u003e gene and the phosphoglycerol dehydrogenase (\u003cem\u003eGpd1\u003c/em\u003e) gene were significantly down-regulated in the ΔOTAbZIP mutant, indicating increased sensitivity to osmotic stress. Previous studies have shown that \u003cem\u003eHog1\u003c/em\u003e is a key regulator of the osmotic stress response, and its down-regulation suggests impaired stress tolerance in the mutant strain [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].The \u003cem\u003eGpd1\u003c/em\u003e gene, which mediates carbohydrate and lipid metabolism, is also down-regulated in the \u003cem\u003eΔOTAbZIP\u003c/em\u003e strain, highlighting its reduced ability to withstand osmotic stress [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eUnder 20 g/L NaCl, the expression of \u003cem\u003eCdc28\u003c/em\u003e, a key protein in the cell cycle, was up-regulated in the mutant strain, suggesting that \u003cem\u003eOTAbZIP\u003c/em\u003e may influence chromatin remodeling and DNA replication processes. In contrast, down-regulation of the \u003cem\u003ePbs2\u003c/em\u003e and \u003cem\u003eHog1\u003c/em\u003e genes in the Δ\u003cem\u003eOTAbZIP\u003c/em\u003e strain suggests that the osmotic stress response pathway is compromised under moderate NaCl stress, although it still allows for adaptation and growth. The altered expression of \u003cem\u003eCtt1\u003c/em\u003e, a catalase involved in oxidative stress protection, further supports the role of \u003cem\u003eOTAbZIP\u003c/em\u003e in regulating cellular defense mechanisms under osmotic stress [\u003cspan additionalcitationids=\"CR45\" citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. It has been reported that high osmolarity conditions increase tyrosine phosphorylation of \u003cem\u003eHog1\u003c/em\u003e to induce peroxidase gene catalase expression to protect cell survival. In contrast, the down-regulated expression of the Ctt1 gene may be due to the increased osmotic pressure sensitivity of the mutant strain Δ\u003cem\u003eOTAbZIP\u003c/em\u003e resulting in the strain itself adapting to the high osmotic pressure environment and thus by up-regulating the expression of the catalase gene (\u003cem\u003eCtt1\u003c/em\u003e) [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Several Studies have shown that the \u003cem\u003eCdc28\u003c/em\u003e-encoding gene is involved in several processes, including chromosome organisation, regulation of cell cycle processes and regulation of metabolic processes of nucleobase-containing compounds. Whereas up-regulation of \u003cem\u003eCdc28\u003c/em\u003e encoding gene indicates that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene may be involved in the regulation of chromatin reorganization during DNA replication, down-regulation of the \u003cem\u003eCdc28\u003c/em\u003e encoding gene for the mutant strain Δ\u003cem\u003eOTAbZIP\u003c/em\u003e may lead to alteration of the meiotic process and DNA replication process, which affects the growth of mycelium [\u003cspan additionalcitationids=\"CR50\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Comparison of WT of \u003cem\u003eA. westerdijkiae\u003c/em\u003e with the mutant strain Δ\u003cem\u003eOTAbZIP\u003c/em\u003e under 100 g/L NaCl showed that the expression of the gene encoding mitogen-activated protein kinase (\u003cem\u003eHog1\u003c/em\u003e) in the HOG-MAPK pathway was down-regulated suggesting that deletion of the \u003cem\u003eOTAbZIP\u003c/em\u003e gene leads to an increased and relatively stable osmotic stress sensitivity in \u003cem\u003eA. westerdijkiae.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eProtein-protein interaction (PPI) network analysis revealed a significant difference in protein connectivity under varying osmolarity conditions. Under 0 g/L NaCl, a relatively low number of interacting proteins was observed, whereas the number of interacting proteins was highest under 20 g/L NaCl, reflecting increased complexity in the stress response. At 100 g/L NaCl, the PPI network showed a decline in protein connectivity, suggesting a shift towards more specialized interactions under extreme osmotic conditions [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFinally, qRT-PCR analysis of the \u003cem\u003eHog1\u003c/em\u003e gene and OTA biosynthesis genes (\u003cem\u003eotaA-D\u003c/em\u003e) revealed that the expression of \u003cem\u003eHog1\u003c/em\u003e was significantly higher in the Δ\u003cem\u003eOTAbZIP\u003c/em\u003e mutant compared to the WT strain under all NaCl conditions. This indicates that the deletion of \u003cem\u003eOTAbZIP\u003c/em\u003e enhances the osmotic stress response, potentially compensating for the absence of OTA production. The expression of the OTA biosynthesis genes in the Δ\u003cem\u003eOTAbZIP\u003c/em\u003e strain was significantly lower than in the WT strain, confirming that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene is essential for OTA production under osmotic stress conditions [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. It indicates that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene significantly affects the OTA biosynthesis genes ota (\u003cem\u003eA-D\u003c/em\u003e), resulting in the inability of the mutant strain to produce OTA. Wang et al. reviewed the regulatory mechanisms of OTA and the role of biosynthetic genes[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Whereas \u003cem\u003eotaD\u003c/em\u003e gene expression did not show a correlation with OTA production, the significant up-regulation of \u003cem\u003eotaD\u003c/em\u003e gene expression under 100 g/L NaCl may be involved in the OTA chlorination step to maintain partial intracellular Cl\u003csup\u003e\u0026minus;\u003c/sup\u003e homeostasis. Our study highlights the critical role of the \u003cem\u003eOTAbZIP\u003c/em\u003e gene in regulating key metabolic and stress response pathways in \u003cem\u003eA. westerdijkiae\u003c/em\u003e, particularly under osmotic stress. The findings from this work provide new insights into the molecular mechanisms underlying OTA biosynthesis and stress tolerance, which may contribute to future strategies for controlling fungal growth and mycotoxin production in agricultural settings\u003c/p\u003e"},{"header":"4. Conclusion","content":"\u003cp\u003eIn this study, RNA-Seq was used to uncover transcriptional changes and key pathways in WT and \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 strains. The \u003cem\u003eOTAbZIP\u003c/em\u003e gene was found to be involved in nucleoside metabolism, glycosyl complex metabolism, and DNA replication under 0 g/L NaCl conditions. At 20 g/L NaCl, \u003cem\u003eOTAbZIP\u003c/em\u003e played a role in sugar and pyruvate metabolism, DNA replication, ribosomal function, and arginine and proline metabolism. Under 100 g/L NaCl conditions, \u003cem\u003eOTAbZIP\u003c/em\u003e influenced glyoxylate and dicarboxylate metabolism, oxidative phosphorylation, and the biosynthesis of unsaturated fatty acids, potentially affecting the growth and OTA production of \u003cem\u003eA. westerdijkiae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, analysis of differential genes in the HOG pathway under varying osmotic pressures, along with qRT-PCR validation of OTA biosynthesis genes (\u003cem\u003eotaA\u003c/em\u003e-\u003cem\u003eD\u003c/em\u003e), revealed that deletion of the \u003cem\u003eOTAbZIP\u003c/em\u003e gene significantly reduced OTA biosynthesis in the \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant strain. Moreover, the expression of \u003cem\u003eHog1\u003c/em\u003e in the \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant was higher than in the WT strain, suggesting increased sensitivity to osmotic stress in the \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant. In conclusion, this study provides valuable insights into the role of \u003cem\u003eOTAbZIP\u003c/em\u003e in the growth and mycotoxin production of \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1, laying a theoretical foundation for strategies aimed at reducing OTA contamination in food.\u003c/p\u003e"},{"header":"5 Materials and Methods","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e5.1 Strains and culture medium\u003c/h2\u003e \u003cp\u003eThe WT OTA-producing strain \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 (PRJNA264608), isolated from agricultural soil obtained from the Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing and the \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 strain [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] constructed by knocking out the \u003cem\u003eOTAbZIP\u003c/em\u003e gene in our laboratory were inoculated into potato dextrose agar (PDA) medium. Both strains were incubated in the dark at 28\u0026deg;C for 7 days. Spores were collected using a sterile cotton swab, resuspended in sterile water and the spore concentration was adjusted to 10\u003csup\u003e7\u003c/sup\u003e spores /mL. Spore suspensions of both the strains were preserved at \u0026minus;\u0026thinsp;80\u0026deg;C with 15% glycerol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e5.2 Total RNA extraction, library construction and Illumina sequencing\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e5.2.1 Sample Preparation and RNA Extraction\u003c/h2\u003e \u003cp\u003eA 100 \u0026micro;L spore suspension of both the WT and \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant strains of \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 was inoculated onto PDA medium and incubated in the dark at 28\u0026deg;C for 72 hours (h). After incubation, 100 mL of PDB medium with NaCl concentrations of 0, 20, and 100 g/L was prepared and sterilized. Each condition was replicated three times. Fresh mycelium from each strain was inoculated into the prepared PDB medium and incubated for 72 h in the dark at 28\u0026deg;C and 160 rpm. After incubation, fungal spores were collected by transferring the cultures to 100 mL centrifuge tubes and centrifuging at 10,000 rpm for 5 min. The supernatant was discarded, and the fungal spores were retained, filtered, and labeled for subsequent total RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e5.2.2. Library Construction and Sequencing:\u003c/h2\u003e \u003cp\u003eThe mRNA with a polyA tail was enriched using Oligo (dT) magnetic beads. The enriched mRNA was then fragmented using divalent cations in NEB Fragmentation Buffer following the NEB standard protocol[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. First-strand cDNA was synthesized using M-MuLV reverse transcriptase, with fragmented mRNA as the template and random oligonucleotides as primers. The RNA strand was then degraded with RNaseH, and the second strand of cDNA was synthesized using dNTPs and DNA polymerase I. The resulting double-stranded cDNA was purified, end-repaired, A-tailed, and ligated to sequencing adapters. Subsequently, cDNA fragments around 250\u0026ndash;300 bp were selected using AMPure XP beads, followed by PCR amplification. The PCR products were purified again using AMPure XP beads to obtain the final libraries [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The kit used for library construction was NEBNext\u0026reg; Ultra\u0026trade; RNA Library Prep Kit for Illumina\u0026reg;. The libraries were quantified preliminarily using a Qubit 2.0 Fluorometer and diluted to a concentration of 1.5 ng/\u0026micro;L. The insert size was assessed using an Agilent 2100 bioanalyzer, and once the size met expectations, the effective concentration of the library was determined by qRT-PCR (ensuring it was above 2 nM) to confirm library quality.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e5.3 GO functional annotation of DEGs and KEGG pathway differential gene analysis\u003c/h2\u003e \u003cp\u003eGO functional enrichment analysis of differentially expressed genes (DEGs) was performed using the cluster Profiler software (3.8.1ver.) [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. This enrichment analysis is based on the hypergeometric distribution principle. The differential gene set consists of genes identified as differentially expressed and annotated in the GO database. In contrast, the background gene set includes all genes analyzed for differential significance and annotated in the GO database. A padj value of less than 0.05 was used as the threshold for significant enrichment. The 30 most significant GO Terms were selected for visualization in histograms; if fewer than 30 were identified, all were displayed. Histograms were categorized according to the three major GO categories: biological processes, cellular components, and molecular functions, along with the classification of up-and down-regulated differential genes.\u003c/p\u003e \u003cp\u003eKEGG pathway enrichment analysis was conducted on the differentially expressed genes of the \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 strain and \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant \u003cem\u003eA. westerdijkiae\u003c/em\u003e strain under three conditions (0, 20, 100 g/L) of NaCl concertation. The analysis was performed using Cluster Profiler software (3.8.1ver.), with a significant threshold of padj less than 0.05. From the KEGG pathway enrichment results, the top 30 significant pathways were selected to create a scatterplot representation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e5.4 Differential Gene Protein Interaction Network Analysis\u003c/h2\u003e \u003cp\u003eThe interactions in the STRING protein interaction database were applied for the analysis of differential gene protein interaction networks. For species included in the database, the interactions of the target gene set were directly extracted from the database to construct the network; for species not included in the database, we firstly applied blastx comparison of the sequences in the target gene set to the protein sequences of the reference species included in the STRING database and constructed the interactions network by using the protein interactions of this reference species on the comparison. Visual editing was performed using Cytoscape software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e5.5 Validation of RNA-Seq analysis by RT-qPCR\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eThe WT and Δ\u003cem\u003eOTAbZIP\u003c/em\u003e mutant of \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 strains were inoculated into PDB medium supplemented with NaCl at concentrations of 0, 20, and 100 g/L. Cultures were incubated at 28\u0026deg;C and 180 rpm for 3 days post-inoculation (dpi). Equal amounts of mycelium were collected from each culture and homogenized in 1 mL Trizol reagent. Samples were incubated at room temperature for 5 min to ensure complete lysis. The homogenates were centrifuged at 12,000 rpm and 4\u0026deg;C for 5 min, and the supernatants were transferred to new 1.5 mL centrifuge tubes. Chloroform (1:5 v/v relative to Trizol) was added, followed by gentle inversion for 30 seconds. Samples were left at room temperature for 5 min to separate phases without agitation to minimize genomic DNA shearing. The upper aqueous phase was carefully transferred to a new tube, mixed with isopropanol (0.5\u0026ndash;1\u0026times; the volume of Trizol), and incubated at room temperature for 10 min. RNA was pelleted by centrifugation at 12,000 rpm and 4\u0026deg;C for 10 min.\u003c/p\u003e \u003cp\u003eThe RNA pellet was washed with 75% ethanol (same volume as Trizol), centrifuged at 7,500 rpm and 4\u0026deg;C for 5 minutes, and air-dried or vacuum-dried for 5\u0026ndash;10 minutes. The RNA was resuspended in an appropriate volume of DEPC-treated water. RNA integrity was confirmed by electrophoresis, and concentrations were measured using a Quawell Q3000 ultra-micro UV spectrophotometer (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). To analyze gene expression, 1 \u0026micro;g of RNA was reverse-transcribed into cDNA using M-MLV reverse transcriptase (Life Technologies, Milan, Italy). Expression levels of OTA synthesis genes (\u003cem\u003eotaA\u003c/em\u003e, \u003cem\u003eotaB\u003c/em\u003e, \u003cem\u003eotaC\u003c/em\u003e, and \u003cem\u003eotaD\u003c/em\u003e) and \u003cem\u003eHog1\u003c/em\u003e were quantified using a Real-Time PCR system with primers listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Amplification conditions were initial denaturation at 95\u0026deg;C for 3 minutes, followed by 35 cycles of 95\u0026deg;C for 10 seconds, 60\u0026deg;C for 45 seconds, and a final extension at 72\u0026deg;C for 7 minutes. Relative expression levels were calculated using the 2-ΔΔCT method with CFX Manager software (Bio-Rad Laboratories) [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. Each treatment was performed in three biological replicates.\u003c/p\u003e\u003cp\u003e\u003cimg 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height=\"554\" width=\"581\"\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRNA concentrations after extraction measured by Quawell-Q3000 ultra-micro UV spectrophotometer\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e260/280\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eConcentration\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT_0 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e56.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔ\u003cem\u003eOTAbZIP\u003c/em\u003e_0 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e325.70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT_20 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e120.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔ\u003cem\u003eOTAbZIP\u003c/em\u003e_20 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e368.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eWT_100 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e397.40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eΔ\u003cem\u003eOTAbZIP\u003c/em\u003e_20 g/L NaCl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eRNA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e1.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1135.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflict of interest:\u003c/h2\u003e \u003cp\u003eAll authors have no conflict of interest and agree to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eThis research was funded by National Key Research and Development Program of China (2022YFE0139500).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, Y.M., M.Z. and Y.L; methodology, Y.M., T.A and M.Z.; formal analysis, G.T., Y.Y and Y.D.; validation, Y.M, T.A. and M.L.; writing\u0026mdash;original draft preparation, M.Z., T.A. and Y.M.; writing\u0026mdash;review and editing, T. A and W.Y; project administration, T.A. and Y.L.; funding acquisition, Y.L.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eXing F, Liu Y, Wang G. Mechanisms of ochratoxin A production in food and strategies for contamination prevention and control. Journal of Food Science and Technology. 2023;41:26\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBattilani P, Magan N, Logrieco A. European research on ochratoxin A in grapes and wine. International Journal of Food Microbiology. 2006;111:S2\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDing X, Liu K, Lu Y, Gong G. Morphological, transcriptional, and metabolic analyses of osmotic-adapted mechanisms of the halophilic Aspergillus montevidensis ZYD4 under hypersaline conditions. Appl Microbiol Biotechnol. 2019;103:3829\u0026ndash;46.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang SA, Rhee KH, Yoo HJ, Pyo MC, Lee K-W. 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Toxins. 2022;14:693.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang G, Li Y, Yang B, Li E, Wu W, Si P, et al. \u003cem\u003eAwAreA\u003c/em\u003e Regulates Morphological Development, Ochratoxin A Production, and Fungal Pathogenicity of Food Spoilage Fungus \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e Revealed by an Efficient Gene Targeting System. Front Microbiol. 2022;13:857726.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSi P, Wang G, Wu W, Hussain S, Guo L, Wu W, et al. SakA Regulates Morphological Development, Ochratoxin A Biosynthesis and Pathogenicity of \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e and the Response to Different Environmental Stresses. Toxins. 2023;15:292.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Liu F, Pei J, Yan H, Wang Y. 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ResearchGate [Internet]. 2024 [cited 2024 Dec 2].\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei J, Liu L, Yuan X, Wang D, Wang X, Bi W, et al. Transcriptome Analysis Reveals the Putative Polyketide Synthase Gene Involved in Hispidin Biosynthesis in \u003cem\u003eSanghuangporus sanghuang\u003c/em\u003e. Mycobiology. 2023;51:360\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Wang L, Liu F, Wang Q, Selvaraj JN, Xing F, et al. Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms. Toxins (Basel). 2016;8:83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMa Y, Li M, Ahmad T, Deng Y, Zhuang M, Tan G, et al. Impact of OTAbZIP on Ochratoxin A production, mycelium growth and pathogenicity of Aspergillus westerdijkiae under water activity stress. 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Targeting a polyketide synthase gene for Aspergillus carbonarius quantification and ochratoxin A assessment in grapes using real-time PCR. Int J Food Microbiol. 2007;115:313\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"OTA, osmotic stress, Aspergillus westerdijkiae, OTAbZIP, gene knockout, HOG-MAPK pathway","lastPublishedDoi":"10.21203/rs.3.rs-5792788/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5792788/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOchratoxins are toxins primarily produced by fungi from the \u003cem\u003eAspergillus\u003c/em\u003e and \u003cem\u003ePenicillium\u003c/em\u003e genera. Among these, Ochratoxin A (OTA) is the most toxic and exhibits strong carcinogenic effects. Reducing food contamination by OTA is a critical global challenge. Osmotic pressure is an important environmental factor that regulates various metabolic pathways in \u003cem\u003eAspergillus westerdijkiae\u003c/em\u003e. It particularly affects the HOG pathway, which controls fungal mycelium growth and OTA production. The \u003cem\u003eOTAbZIP\u003c/em\u003e gene is a key transcription factor and receptor in \u003cem\u003eA. westerdijkiae\u003c/em\u003e. It plays a vital role in responding to osmotic pressure and regulating the expression of OTA biosynthesis genes (\u003cem\u003eotaA-D\u003c/em\u003e). This study explored the role of \u003cem\u003eOTAbZIP\u003c/em\u003e in fungal growth and OTA production under different osmotic conditions using RNA-Seq and RT-qPCR analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTranscriptomic analysis showed that the \u003cem\u003eOTAbZIP\u003c/em\u003e gene influences several pathways. These include DNA replication, sugar metabolism, ribosome function, and arginine and proline metabolism at NaCl concentrations of 0, 20, and 100 g/L. Genes in the high osmolarity glycerol, mitogen-activated protein kinase (MAPK HOG) pathway, such as \u003cem\u003eHog1\u003c/em\u003e, \u003cem\u003eGpd1\u003c/em\u003e, \u003cem\u003eCdc28\u003c/em\u003e, and \u003cem\u003eCtt1\u003c/em\u003e were affected. The \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant strain exhibited increased sensitivity to osmotic stress. RT-qPCR results confirmed that OTA biosynthesis gene expression was significantly lower in the \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutant strain than in the wild-type (WT) strain of \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1. However, the \u003cem\u003eΔOTAbZIP\u003c/em\u003e mutation did not affect the activation of the \u003cem\u003eHog1\u003c/em\u003e gene.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study highlights the role of the \u003cem\u003eOTAbZIP\u003c/em\u003e gene in regulating metabolic pathways linked to growth and OTA production in \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1. These findings enhance our understanding of the gene's functions of \u003cem\u003eA. westerdijkiae\u003c/em\u003e fc-1 under various somatic pressures. They also offer insights for developing strategies to control OTA contamination in food and feed.\u003c/p\u003e","manuscriptTitle":"Role of the OTAbZIP Gene in Regulating Growth and OTA Production in Aspergillus westerdijkiae fc-1 Under Osmotic Stress","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-13 16:19:07","doi":"10.21203/rs.3.rs-5792788/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d98ec789-5447-4a07-8b3b-6a32cb27ba58","owner":[],"postedDate":"January 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-02-16T16:08:12+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-13 16:19:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5792788","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5792788","identity":"rs-5792788","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}