3.1 Sequence analysis and expression patterns of AoZC9
To delineate the expression profile of the AoZC9 gene during distinct growth phases of A. oryzae and across a gradient of salt concentrations, quantitative real-time PCR (qRT-PCR) analysis was conducted. Samples were collected at 24, 48, and 72 hours, representing the adaptive, logarithmic, and stationary phases, respectively. The mRNA levels were assessed under salt concentrations of 0%, 5%, 10%, and 15%. As depicted in Figure 1A, AoZC9 exhibited a significant upregulation from the early to the later growth phases and displayed a notable downregulation under increased salinity, suggesting a stage-specific and stress-responsive regulatory function.
The open reading frame (ORF) of AoZC9, which encodes a protein comprising 178 amino acids (aa), was successfully cloned utilizing a set of specifically designed primers. Subsequent domain analysis revealed that the AoZC9 protein features two identical conserved domains, designated as fungal-TF-MHR. The first of these domains is situated within the region spanning from residue 1 to residue 297, as depicted in Figure 1B. To further elucidate the evolutionary connections between the AoZC9 protein from various Aspergillus species, a phylogenetic tree was constructed using a maximum likelihood approach based on aligned amino acid sequences. The phylogenetic analysis, illustrated in Figure 1C, suggests a close evolutionary relationship between AoZC9 and the protein XP_002377833 from A. flavus.
3.2 AoZC9-overexpression diminishes salt tolerance and fatty acid profiles in Saccharomyces cerevisiae
The impact of AoZC9 overexpression on salt tolerance in S. cerevisiae was assessed by monitoring the growth kinetics of yeast strains under salt stress conditions. Optical density (OD) measurements at 12-, 24-, and 36-hours post-treatment revealed that strains overexpressing AoZC9 displayed significantly reduced growth rates compared to the wild-type when exposed to salt concentrations, as evidenced by diminished OD values (Fig. 2A-C). This indicates that the presence of AoZC9 negatively affects the ability of yeast to tolerate saline conditions. Furthermore, a serial dilution assay confirmed the growth inhibition in transgenic strains, with a more pronounced suppression at 10% NaCl compared to the wild-type (Fig. 2D).
To delve into the physiological effects of AoZC9 overexpression, we analyzed the fatty acid composition of the yeast strains using gas chromatography. The results showed a significant decrease in UFA levels, including C18:1n9, C18:2n6, C18:3n3, and C20:2n6, in strains overexpressing AoZC9, while the levels of saturated fatty acids, such as palmitic and stearic acids, were relatively increased (Fig. 2E). This alteration in fatty acid profiles suggests a potential mechanism by which AoZC9 overexpression impairs salt tolerance in yeast.
3.3 AoZC9-overexpression diminishes salt tolerance and fatty acid profiles in Aspergillus oryzae
Under salt stress, WT, AoZC9-overexpressing, and RNAi-AoZC9 strains of A. oryzae exhibited some degree of growth inhibition, yet the extent of this inhibition varied significantly among them. Notably, as the salt concentration increased, the overexpression of AoZC9 led to a more pronounced growth suppression compared to the WT, particularly as the salt concentration increased to 15% (Fig. 3).
In contrast, the RNAi strains, with reduced AoZC9 expression, showed less growth inhibition under salt stress than the WT, indicating a potential adaptive advantage. Strikingly, as the salt concentration escalated to 15%, the RNAi strains not only maintained higher spore density and dry biomass but also exhibited approximately twice the spore count and biomass compared to the WT. This enhancement in growth parameters was also reflected in the mycelial diameter, which was notably larger in the RNAi strains under high salt conditions. RNAi strains indicates that the downregulation of AoZC9 may activate alternative pathways or mechanisms that improve the ability of strains to withstand salt stress (Fig. 3). These contrasting phenotypes between the overexpressing and RNAi strains underscore the complex regulatory role of AoZC9 in the adaptation to salt stress.
3.4 Reduction in unsaturated fatty acid content and FAD2 expression
Prior research has established a link between salt tolerance and fatty acid composition, prompting an investigation into the effects of the Ao9639 gene on these parameters in A. oryzae. Our findings demonstrate that the overexpression of the AoZC9 gene is correlated with a decrease in the expression of the FAD2 gene, which encodes a key enzyme in the conversion of saturated to UFAs. This downregulation of FAD2 expression is further reflected in the reduced levels of UFAs observed in the overexpressing strains. In contrast, strains with RNAi targeting AoZC9 show an upregulation of FAD2 expression and a corresponding increase in the intracellular content of UFAs, suggesting a specific regulatory role of AoZC9 on the unsaturated fatty acid pathway. These findings provide evidence for the negative regulatory relationship between AoZC9 and FAD2, with implications for the control of fatty acid composition in A. oryzae. The statistical analysis, represented by the error bars in the Figure 4, confirms the reproducibility and significance of the observed differences.
3.5 Transcriptome overview
To further elucidate the molecular mechanisms underlying the function of AoZC9, we conducted a transcriptome analysis on the overexpressing and RNAi of AoZC9 in A. oryzae strains. The sequencing data from this analysis demonstrate a high mapping efficiency across all samples. In the WT, a total of 29,986,270 reads were generated, with 27,140,887 (90.51%) successfully mapped to the reference genome. Of these mapped reads, 27,058,859 (90.24%) were uniquely mapped, while a small fraction, 82,028 (0.27%), mapped to multiple locations (Table 1).
For the overexpression-AoZC9 strain, the total read count was higher at 41,514,850. These reads showed a mapping efficiency of 95.71%, with 39,733,790 reads aligned to the genome. The unique mapping rate was slightly higher than in the WT at 95.21%, corresponding to 39,525,625 reads. The number of multiple mappings was also higher in the overexpressing strain, with 208,165 reads (0.50%) aligning to multiple sites. The RNAi-AoZC9 strain exhibited the highest mapping efficiency among the three, with 96.38% of the total 40,872,100 reads mapped. Uniquely mapped reads were 39,247,380 (96.02%), and the multiple mappings were the lowest among the strains at 145,552 (0.36%).
3.6 Comparative differentially expressed gene analysis in Aspergillus oryzae
The transcriptome analysis of the WT, overexpression-AoZC9, and RNAi-AoZC9 strains of A. oryzae under normal conditions has yielded a distinct set of DEGs. The comparison between these strains was conducted to understand the impact of AoZC9 gene expression modulation on the global transcriptional profile of the organism.
In the comparison between the WT and the overexpression-AoZC9 strains, a total of 6,912 DEGs were identified, with 3,101 genes upregulated and 3,811 genes downregulated (Table2). This significant shift in gene expression underscores the substantial influence of overexpression-AoZC9 on the cellular transcriptome. When contrasting the WT with the RNAi-AoZC9 strains, 5,090 DEGs were observed, comprising 2,553 upregulated and 2,537 downregulated genes. This indicates that the RNAi-mediated AoZC9 also results in a considerable change in gene expression, suggesting a critical role for this gene in regulating the transcriptional state of the cell. The direct comparison between the overexpression-AoZC9 and the RNAi-AoZC9 strains revealed 5,521 DEGs, with 3,222 genes upregulated and 2,299 genes downregulated. This comparison highlights the contrasting transcriptional responses elicited by the overexpression and RNAi of AoZC9. Notably, 1725 DEGs were consistently observed across all three comparisons, indicating that these genes may be central to the regulatory network influenced by AoZC9 (Fig. 5).
3.7 KEGG pathway enrichment analysis of differentially expressed genes
To delve deeper into the functions of the DEGs, we mapped them onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and conducted an enrichment analysis 23. Among the DEGs between the WT and the overexpression-AoZC9 strains, the pathways with the highest number of DEGs and the smallest q-values were identified as sphingolipid metabolism (ko00600), biosynthesis of UFAs (ko01040), mitogen-activated protein kinase (MAPK) signaling pathway (ko04011), steroid biosynthesis (ko00100), and ATP-binding cassette (ABC) transporters (ko02010) (Table 3).
When comparing the WT to the RNAi-AoZC9, the pathways with the lowest q-values were glutathione (GSH) metabolism (ko00480), biosynthesis of UFAs (ko01040), MAPK signaling pathway (ko04011), ABC transporters (ko02010), and pyruvate metabolism (ko00620).
In the comparison between the overexpression and RNAi-AoZC9 strains, besides the pathways mentioned above, the DEGs also participated in pyruvate metabolism (ko00620) and glycine, serine, and threonine metabolism (ko00260), with the smallest q-values observed. Notably, the top five pathways are all related to the biosynthesis of UFAs, further suggesting that the transcription factor may influence salt tolerance by regulating the content of UFAs.