The Deletion of LeuRS Revealed Its Important Roles in Osmotic Stress Tolerance, Amino Acid and Sugar Metabolism, and the Reproduction Process of Aspergillus montevidensis

Aspergillus montevidensis is an important domesticated fungus that has been applied to produce many traditional fermented foods under high osmotic conditions. However, the detailed mechanisms of tolerance to osmotic stress remain largely unknown. Here, we construct a target-deleted strain (ΔLeuRS) of A. montevidensis and found that the ΔLeuRS mutants grew slowly and suppressed the development of the cleistothecium compared to the wide-type strains (WT) under salt-stressed and non-stressed conditions. Furthermore, differentially expressed genes (p < 0.001) governed by LeuRS were involved in salt tolerance, ABC transporter, amino acid metabolism, sugar metabolism, and the reproduction process. The ΔLeuRS strains compared to WT strains under short- and long-term salinity stress especially altered accumulation levels of metabolites, such as amino acids and derivatives, carbohydrates, organic acids, and fatty acids. This study provides new insights into the underlying mechanisms of salinity tolerance and lays a foundation for flavor improvement of foods fermented with A. montevidensis.


Introduction
The filamentous fungi Aspergillus is a complex group of ascomycetes comprising about 340 officially recognized species, many of which are widely used in food fermentation [1].Among Aspergillus species, A. montevidensis is an important domesticated fungus that has been used to produce or isolate from fermented and ripened katsuobushi [2]. A. amstelodami (the anamorph of A. montevidensis) is commonly isolated from meju, a brick of fermented dried soybeans, and frequently occurs in fermented cocoa beans [3,4].Furthermore, these species dominate the microbial community involved in post-fermented Chinese dark teas, including Pu-erh tea, Fu brick tea, and Liupao tea [5][6][7].During the production processes of these foods, high osmolarity conditions enhance the dominance of A. montevidensis, which can result in improved quality and taste of fermented foods.Therefore, it is of great significance to understand the osmophilic mechanism of A. montevidensis.
We previously found that A. montevidensis is highly dependent on the fine regulation of morphological, transcriptional, and metabolic responses to manage osmotic stress tolerance [8].Under salinity stress, A. montevidensis widely developed yellow fruiting bodies (cleistothecia), which are known as 'golden flowers' in Chinese brick tea, and hypha growth was obviously promoted [9].Salinity-induced mycelia significantly expressed hundreds of genes that controlled cellular processes, such as oxidative stress response, amino acid transport and metabolism, glycolysis and TCA cycles, and fatty acid β-oxidation, for the intracellular accumulation of a variety of amino acids, soluble sugars, and fatty acids.Among these significantly expressed genes, a unigene encoding leucyl-tRNA synthetase (LeuRS), The wide-type A. montevidensis (no: CGMCC 3.15762) used in this study was maintained routinely on yeast peptone dextrose agar slants (YPD: 1% yeast extract, 2% tryptone, 2% glucose, 1.5% agar, pH 6.5), supplemented with the final NaCl concentration (1.5 M).The morphological and growth rates of the WT and ∆LeuRS mutants were examined on YPD media with 0 M and 1.5 M NaCl at 28 • C. The WT and mutants were statically cultured in YPD liquid medium with 1.5 M NaCl at 28 • C for 14 days (long-term salt stress, WT_LS and ∆LeuRS_LS).These strains were also grown in YPD liquid medium without salt at 28 • C for 14 days and then stressed by the addition of salt (1.5 M) for 1 h (short-term salt stress, WT_SS and ∆LeuRS_SS).Control samples (WT_C and ∆LeuRS_C) were obtained from 14-day cultures untreated with salt.Salt-treated and untreated cultures were washed twice with a pre-chilled PBS solution.Mycelia were collected using centrifugation at 4 • C, 12,000 rpm for 10 min, and used in the examination of transcriptome, metabolome, and antioxidant enzyme activity (catalase, CAT; superoxide dismutase, SOD) [19].

Transcriptome Sequencing and Data Analysis
RNA extraction and library preparation of salt-treated and untreated cultures were performed as described by Ding et al. [8].The cDNA libraries were sequenced on an Illumina Novaseq 6000 platform and 150-bp paired-end reads were generated.The raw data were processed by removing adaptor sequences, low quality reads, and reads with unknown nucleotides > 5%.High-quality read assembly was performed using Trinity software (v2.4.0) [22].With three biological replicates, transcriptomic sequencing generated 383,214,494 and 399,020,304 clean reads for the three comparison groups (18 libraries).Clean reads were assigned to the reference genome (accesssion number JAJFZZ000000000) [20].The RPKM (reads per kb per million reads) of each unigene was calculated to analyze the expression level of genes between samples.Genes with a false discovery rate (FDR) < 0.001 and |log2 ratio| ≥ 1 were identified as differentially expressed genes (DEGs) [23].Principal component analysis (PCA) was performed using the R package (version 4.0.0).DEG enrichment analysis was performed in different databases using BLASTx, including NCBI nonredundant protein (NR), Gene Ontology (GO), Swissprot, KOG, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) with E < 10 −5 .The FPKM values of DEGs were used in a heatmap analysis.The biochemical pathways of DEGs were further implemented by KEGG mapping using the cluster Profiler (v3.4.4) [24].

UHPLC-Q-TOF/MS of Metabolites and Data Analysis
The salinity-treated and non-treated freeze-dried fungal mycelia (0.1 g) of the WT and the mutant strains (∆LeuRS) were extracted with methanol-acetonitrile-water (2:2:1, v/v), which contained internal standard mixtures labeled with isotopes.The samples were vortexed, sonicated in an ice water bath for 4 min, incubated at −40 • C for 1 h, and then centrifuged at 4 • C for 15 min (12,000 rpm) to collect the supernatants.The extraction process was repeated three times.The supernatants were pooled and filtered through a 0.22-µm filter.Meanwhile, a quality control (QC) sample was prepared by mixing equal volumes of each extraction, and was injected to monitor the stability of the system.Metabolites were analyzed using an UHPLC system (Vanquish, Thermo Fisher Scientific, Pleasanton, CA, USA) with a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 µm) coupled with a Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo Fisher).The mobile phase consisted of water (pH 9.75) (containing 25 mmol/L ammonium acetate and 25 mmol/L ammonia hydroxide) and acetonitrile.The autosampler temperature was 4 • C and the injection volume was 3 µL.The elution gradient was carried out as follows: 0 min, 5% B; 3 min, 20% B; 9 min, 95% B; 13 min, 95% B; 13.1 min, 5% B; and 16 min, 5% B. A mass scan range of m/z 70-1000 was performed for full-scan analysis.The ESI source conditions were established as follows: sheath gas flow rate 30 arb, aux gas flow rate 25 arb, capillary temperature 350 • C, full MS resolution 60,000, collision energy 10/30/60, and spray voltage 3.6 (positive) or −3.2 kV (negative), respectively.The raw mass data were converted into mzXML format using ProteoWizard software (v3.0.9134).XCMS software (v3.7.1) was applied for peak extraction, peak alignment, and peak matching.Metabolites were then annotated using BiotreeDB (v2.1) software.

Statistical Analysis
All experiments were conducted in independent triplicates, and data are reported as the mean value ± SD (standard deviation).Student's t-test was performed to examine the significant differences in metabolites between groups (p < 0.05) by using SPSS 26.0 software (SPSS Inc., Chicago, IL, USA).Differential metabolites with absolute log2FC ≥ 1 and p < 0.05 were selected for further analysis.Orthogonal projections to latent structures discriminant analysis (OPLS-DA) using SIMCA 14.1 [25] were conducted to screen characteristic components contributing to group discrimination.Differential metabolites were selected by the statistically significant variable importance threshold in projection values ≥1 and p-values < 0.05.Significantly different data sets in transcriptomic and metabolomics analysis were used to generate metabolite-transcript pathways by KEGG mapping.

Accession Number(s)
The raw sequence data have been submitted to the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under BioSample accessions: SRR24694851 to SRR24694851.

LeuRS Influences Mycelial
Growth, Cleistothecium Formation, and Stress Tolerance of A. montevidensis ∆LeuRS mutants were verified using PCR amplification and sequencing (Figure S1).Mycelial growth showed a morphological difference between the WT strain and the LeuRS mutant on the YPD medium supplemented with or without NaCl.The WT strain had yellow colonies, while the ∆LeuRS colonies were white under salinity or non-salt stress for 14 days (Figure 1).The WT strain also produced a large number of yellow-colored spherical cleistothecia (Figure 1b,c), while the formation of cleistothecium was strongly inhibited in the LeuRS mutant (Figure 1e,f).The deletion of LeuRS resulted in a significant reduction (p < 0.001) of the growth rates of the hyphal compared to WT strains under salinity or nonsalt conditions (Figure 1k).Salt stress causes oxidative stress in living cells [26].Therefore, we examined the antioxidative activities of the LeuRS mutant and the WT strain.CAT activity in the ∆LeuRS cultures induced with or without NaCl was significantly (p < 0.05) lower than in WT, and SOD activity in mutant samples suffering from long-term salinity stress was obviously lower compared to the corresponding WT group (Figure 1l).These results indicated that LeuRS plays a crucial role in the regulation of mycelial growth, antioxidative enzyme activities, and sexual development of A. montevidensis.

Transcriptome Profiles of the LeuRS Mutant and the WT Strain
We matched unigene sequences with the NR, Swiss-prot, KEGG, and KOG databases using blastx (E < 10 −5 ).Of these, a total of 7165 unigenes were functionally annotated.PCA indicated that all biological replicates were grouped together, and PC1 and PC2 captured 60% of the variance in the data.This represents the difference in gene expression profiles between the WT strain and the ∆LeuRS mutant (Figure 2a).We identified significantly differentially expressed unigenes (DEGs) using FDR < 0.001 and the |log 2 ratio| ≥ 1 as the threshold.Among the genes significantly expressed up, there were 82 DEGs in the ∆LeuRS_SS/∆LeuRS_C and 1107 in the ∆LeuRS_LS/∆LeuRS_C, with 50 DEGs shared by these two groups (Figure 2b).There were 153 DEGs in the WT_SS/WT_C and 881 in the WT_LS/WT_C, with 62 co-occurred DEGs (Figure 2c).Among the significantly down-expressed genes, there were 79 DEGs in the ∆LeuRS_SS/∆LeuRS_C and 796 in the ∆LeuRS_LS/∆LeuRS_C, with 51 common DEGs (Figure 2d).There were 576 DEGs in the WT_SS/WT_C and 1070 in the WT_LS/WT_C, with 370 common DEGs (Figure 2e).Furthermore, more DEGs responded to long-term saline conditions than to short-term treatment, but the majority of genes in response to short-term treatment also responded to long-term salt stress (Figure 2b,d,e).Our results showed that LeuRS was of significance in mediating gene expression in A. montevidensis in response to change in salinity.

Transcriptome Profiles of the LeuRS Mutant and the WT Strain
We matched unigene sequences with the NR, Swiss-prot, KEGG, and KOG databases using blastx (E < 10 −5 ).Of these, a total of 7165 unigenes were functionally annotated.PCA indicated that all biological replicates were grouped together, and PC1 and PC2 captured WT_SS/WT_C and 1070 in the WT_LS/WT_C, with 370 common DEGs (Figure 2e).Furthermore, more DEGs responded to long-term saline conditions than to short-term treatment, but the majority of genes in response to short-term treatment also responded to long-term salt stress (Figure 2b,d,e).Our results showed that LeuRS was of significance in mediating gene expression in A. montevidensis in response to change in salinity.

Analysis of DEGs
KEGG pathway analysis of DEGs revealed that more metabolic processes in ∆LeuRS and WT were influenced by long-term salt stress than short-term saline shock (Figure 3).For example, many DEGs up-regulated (p < 0.05) in ∆LeuRS_LS/∆LeuRS_C participated in 13 pathways (fructose and mannose metabolism, tyrosine metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, ABC transporters, etc.).However, a few DEGs were associated with three pathways in ∆LeuRS_SS/∆LeuRS_C.Furthermore, the number of DEGs expressed up, involved in alanine, aspartate, and glutamate metabolism and nitrogen metabolism, increased in ∆LeuRS as stress continued.The DEGs descending in expression were mainly related (p < 0.05) to four KEGG pathways (biosynthesis of amino acids, peroxisome, butanoate metabolism, etc.) in ∆LeuRS_LS/∆LeuRS_C.Similarly, many DEGs up-regulated in WT_LS/WT_C were strongly enriched in (p < 0.05) ribosome biogenesis, pentose and glucuronate interconversions, and sphingolipid metabolism, while a few genes participated in oxidative phosphorylation in WT_SS/WT_C.And the decreased DEGs in WT_LS/WT_C were enriched (p < 0.05) in 11 pathways, which were indispensable to starch and sucrose metabolism, glycine, serine and threonine metabolism, and arginine and proline metabolism, etc. (Figure 3d).

Analysis of DEGs
KEGG pathway analysis of DEGs revealed that more metabolic processes in ΔLeuRS and WT were influenced by long-term salt stress than short-term saline shock (Figure 3).For example, many DEGs up-regulated (p < 0.05) in ΔLeuRS_LS/ΔLeuRS_C participated in 13 pathways (fructose and mannose metabolism, tyrosine metabolism, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, ABC transporters, etc.).However, a few DEGs were associated with three pathways in ΔLeuRS_SS/ΔLeuRS_C.Furthermore, the number of DEGs expressed up, involved in alanine, aspartate, and glutamate metabolism and nitrogen metabolism, increased in ΔLeuRS as stress continued.The DEGs descending in expression were mainly related (p < 0.05) to four KEGG pathways (biosynthesis of amino acids, peroxisome, butanoate metabolism, etc.) in ΔLeuRS_LS/ΔLeuRS_C.Similarly, many DEGs up-regulated in WT_LS/WT_C were strongly enriched in (p < 0.05) ribosome biogenesis, pentose and glucuronate interconversions, and sphingolipid metabolism, while a few genes participated in oxidative phosphorylation in WT_SS/WT_C.And the decreased DEGs in WT_LS/WT_C were enriched (p < 0.05) in 11 pathways, which were indispensable to starch and sucrose metabolism, glycine, serine and threonine metabolism, and arginine and proline metabolism, etc. (Figure 3d).

DEGs Significantly Related to ABC Transporters, Nitrogen and Carbon Metabolism, and Reproduction
To understand the transcriptional changes of DEGs of ∆LeuRS and WT in response to salt stress, response-specific DEGs were selected for heatmap analysis (Figure 4, Table S1).We found that most DEGs in ∆LeuRS_C, ∆LeuRS_SS, and ∆LeuRS_LS exhibited distinct expression patterns, compared to their transcriptional levels in the corresponding WT samples.For example, the expression two unigenes encoding ABC transporters was dramatically decreased in ∆LeuRS_C and ∆LeuRS_SS and increased in ∆LeuRS_LS, while the reverse trend was shown in WT_C, WT_SS, and WT_LS.Genes homologous to adenylosuccinate synthase, aspartate aminotransferase, and 1-pyrroline-5-carboxylate dehydrogenase (which were involved in alanine, aspartate, and glutamate metabolism) exhibited lower expression levels in mutants than in WT strains with different osmotic conditions.Gene-coding enzymes such as ornithine carbamoyltransferase, glutamate dehydrogenase, arginase, and argininosuccinate lyase, which control arginine biosynthesis, were strongly down-regulated in ∆LeuRS groups, while these had high transcriptional levels in WT.Similar expression patterns of genes were also observed in tryptophan metabolism.Genes related to sugar metabolism and glyoxylate and dicarboxylate metabolism, such as malate dehydrogenase, 6-phosphogluconate dehydrogenase, and glycine hydroxymethyltransferase were increased in expression in the three treatment groups of ∆LeuRS but strongly inhibited in WT.We found that some regulatory factors, which regulate amino acid metabolism, sexual reproduction, and tolerance to osmotic stress, were down-regulated in mutants.For example, several genes encoding Zn (II) 2 Cys6 (C6) transcription factors exhibited lower expression levels in ∆LeuRS against them in WT.Genes (such as MAT1, Ste20, and VeA) involved in sexual reproduction were obviously suppressed in ∆LeuRS compared to those in WT.Similarly, mitogen-activated protein kinase (HOG1) was also negatively regulated in ∆LeuRS.We observed that ∆LeuRS_LS augmented the expression of many genes more than ∆LeuRS_C and ∆LeuRS_SS.In addition, we also compared the expression changes of DEGs in ∆LeuRS with them in WT under the same osmotic stress conditions.We found that most DEGs of ∆LeuRS were down-regulated in expression and that the expression levels of many genes increased as the osmotic conditions continued.Our findings demonstrated that LeuRS mediated many cellular processes (substance transport, amino acid and sugar metabolism, and reproduction) of the fungus under salt stress.

Discussion
A. montevidensis is commonly used in the production of traditional fermented foods [4,7].During fermentation processes, the transcriptional responses of A. montev-idensis to osmotic conditions are necessary for the flavor formation of foods.To reveal the functions of LeuRS (which were strikingly expressed higher in A. montevidensis under osmotic stress), we constructed a knockout mutant of LeuRS and found that the deletion of LeuRS inhibited growth both in the presence and absence of NaCl and deactivated the formation of cleistothecium compared to the WT strain (Figure 1).We also found another LeuRS (which was designated as mLeuRS, encoding a mitochondrial leucyl-tRNA synthetase) in the fungus.The average FPKM values of mLeuRS were higher in ∆LeuRS_C (25.7 FPKM) and ∆LeuRS_SS (24.9 FPKM) than in WT_C (5.0 FPKM) and WT_SS (6.2 FPKM), but lower in ∆LeuRS_LS (5.2 FPKM) compared to WT_LS (11.1 FPKM).This could be the reason why the knockout of LeuRS in the fungus did not cause lethality.Transcriptomic and metabolomic analyses revealed that LeuRS are closely related to ABC transporters, amino acid metabolism, carbohydrate metabolism, and sexual development of A. montevidensis.
Here, we observed that DEGs associated with ATP-binding cassette (ABC) transporters were down-regulated in ∆LeuRS, while they were obviously increased in WT samples (Figure 4, Table S1).Long-term saline induction increased the transcriptional levels of ABC transporters in mutants.Extensive research showed that ABC transporters are evolutionarily conserved integral membrane proteins responsible for the allocation of a wide variety of substrates, including ions, sugars, amino acids, polypeptides, complex lipids, toxic metabolites, and even toxins [27,28].Previous reports also indicated that the specific upregulation of some ABC transporters was involved in cell osmo-stress tolerance [29,30].
Specific regulation of amino acid metabolism is crucial to controlling cell growth and proliferation, and osmotic adjustment of all living organisms [8,31,32].As expected, 34 significant DEGs between ∆LeuRS and WT groups under short-and long-term salt stress were associated with the metabolism pathways of many amino acids (Table S1).We found that many up-expressed genes of ∆LeuRS mutants with short-and long-term saline stresses, compared to controls (∆LeuRS_C), were significantly enriched in four KEGG pathways of amino acid metabolism (alanine, aspartate, and glutamate metabolism, arginine and proline metabolism, and tryptophan metabolism, etc.).In contrast, the decreased expression of DEGs between the WT_LS vs. WT_C groups were mainly enriched in alanine, aspartate, and glutamate metabolism, and arginine and proline metabolism.Furthermore, we observed that ∆LeuRS_LS up-expressed DEGs in mitophagy (Figure 3a), suggesting that the recycling of nutrients to avoid carbon and nitrogen starvation may be an important survival strategy for the fungus with long-term cultivation under osmotic stress.
We found that ∆LeuRS mutants exposed to salt stress increased the expression of genes encoding glutamine synthetase and aspartate carbamoyl transferase, which were associated with alanine, aspartate, and glutamate metabolism.These proteins were up-regulated in salt-induced plants [33,34].However, ∆LeuRS with or without salt significantly inhibited the transcription of gene-encoding enzymes such as aspartate aminotransferase, succinatesemialdehyde dehydrogenase, and 1-pyrroline-5-carboxylate dehydrogenase.Previous reports showed that up-expression of aspartate aminotransferase and 1-pyrroline-5-carboxylate dehydrogenase was important for the survival of plants under saline conditions [33,35].Furthermore, we observed that WT strains under long-term saline conditions increased the levels of some amino acids and organic acids (e.g., DL-phenylalanine, N-(2-furoyl) glycine, and 3-methyladipic acid) (Figure 5), which could contribute to the salt adaptation of the fungus.
Previous studies indicated that the activation of arginine and proline metabolism confers enhanced stress tolerance in different eukaryotes [8,36].However, little is known about the regulatory mechanism of the pathway.Here, we found that DEGs were significantly enriched in arginine and proline metabolism (Figure 4, Table S1).The expression of genes encoding ornithine decarboxylase and the S-adenosylmethionine decarboxylase proenzyme increased in ∆LeuRS_LS and WT_LS, compared to their controls.These genes participated in the biosynthesis of polyamines, which can improve plant tolerance to salinity by scavenging free radicals [37].A gene-encoding D-amino acid oxidase was up-expressed in ∆LeuRS_LS/∆LeuRS_C, which accelerates the process of proline transformation into 1pyrroline-2-carboxylate in proline metabolism.A previous study showed that the increase in D-amino-acid oxidase activity was correlated with the heat tolerance of fungi [38].Interestingly, the expression of all genes (such as carbamoyl-phosphate synthase, arginase, and argininosuccinate synthase) in the ornithine cycle was strongly down-regulated in ∆LeuRS groups, while they maintained high transcriptional levels in WT samples.In addition, two gene-encoding arginase and argininosuccinate synthase in mutants were fully blocked.The deletion of carbamoyl-phosphate synthase in Colletotrichum gloeosporioides led to a slow growth rate and an extreme sensitivity to high osmotic stress [39].Up-regulation of arginase could alleviate damage to plants caused by salinity [40].These suggested that LeuRS could mediate the ornithine cycle in the fungal adaptation to salinity.Moreover, we observed that some genes in tryptophan metabolism also maintained relatively low expression in LeuRS_C and LeuRS_SS, but they were enhanced in LeuRS_LS.A similar case in the plant showed that activation of the phenylalanine, tyrosine, and tryptophan pathways improved osmoprotective ability [41].∆LeuRS under salt stress specially modulated the metabolic flow of some amino acids, which might rescue mutant defects in terms of salt tolerance.
Sugar metabolism has been shown to play a key role in maintaining osmotic homeostasis and intracellular energy balance through carbon partitioning in microbes [42].Here, we observed that many DEGs in ∆LeuRS maintained relatively low expression, which encoded enzymes involved in the glycolysis/glucoseogenesis, citrate cycle, and glyoxylate and dicarboxylate metabolism (Table S1).Hexokinases control the first step of all major pathways of glucose utilization and therefore influence the extent and direction of glucose flux within the cell.The expression of hexokinases promoted tolerance to drought and salt stress in plants [43].Increased expression levels of enolase and malate dehydrogenase in ∆LeuRS could promote the fungus to overcome salt.These genes were also up-regulated in halophytes exposed to hypersaline stress [44,45].In addition, activation of glyoxylate and dicarboxylate metabolism was shown in sesames for tolerance to salinity [46].Mutants under long-term salinity stress markedly increased the expression of genes coding catalase (which catalyzes the dismutation of H 2 O 2 into H 2 O and O 2 ) but almost abolished the expression of (S) -2-hydroxy acid oxidase (which transforms glycolate into H 2 O 2 and glyoxylate), suggesting that the elimination of reactive oxygen species is important for fungal osmoadaptation.And the salt-induced WT samples up-regulated many compatible sugars (e.g., sucrose, D-mannose, and D-mannose 1-phosphate) (Figure 5), suggesting that soluble sugars could act as osmoprotectants in the fungus.
We found that some genes encoding transcription factors (such as the Zn (II) 2 Cys6 (C6) transcription factor, BrlA, and HOG1), maintained relative low expression in ∆LeuRS (Table S1).C6-like transcription factors regulated the metabolism of branched-chain amino acids (leucine, isoleucine, and valine) in A. fumigatus and sexual development in N. crassa [47,48].BrlA, a central regulator, activates the conidial developmental pathway of A. nidulans [13].And activation of the HOG1-mediated pathway promotes osmotic tolerance and influences sexual reproduction of fungi [49,50].In addition, ∆LeuRS varied the expression levels of other reproduction-related genes in aspergilli.The core 'velvet' proteins (VeA, VelB, and LaeA) play a pivotal role in light and dark regulation of sexual development [51].The removal of VeA increased the sensitivities to high osmolarity, oxidation stress, and triggered the production of fungal conidia [52].Collectively, our results suggested that LeuRS modulated the metabolism of some amino acids and fungal reproduction by regulating the activities of relative transcription factors.

Conclusions
We constructed the ∆LeuRS mutant and characterized the cellular function of LeuRS in A. montevidensis under osmolarity conditions.Our results demonstrated that the deletion of LeuRS influenced the expression of multiple genes of the halophilic fungus.We observed that the mutants varied the expression levels of many genes that govern reproduction processes in the fungus (Figure 6a).We found that DEGs of ∆LeuRS mutants and WT strains were significantly enriched in KEGG pathways such as ABC transporters, alanine, aspartate and glutamate metabolism, arginine and proline metabolism, ornithine cycle, and sugar metabolism (Figure 6b).Metabolomic analyses showed that a range of metabolites varied significantly in this pairwise comparison, such as amino acids, sugars, and organic acids.The findings will help reveal the osmoadaptation strategies of A. montevidensis under high saline conditions.Genes marked with red or green color showed that these were up-and down-regulated at different time points.

Figure 2 .
Figure 2. PCA (a) and Venn analysis of genes up-(b,c) and down-expressed (d,e) in ΔLeuRS mutants and WT strains under different saline conditions (C = control, SS = short-term salt stress, LS = longterm salt stress).

Figure 2 .
Figure 2. PCA (a) and Venn analysis of genes up-(b,c) and down-expressed (d,e) in ∆LeuRS mutants and WT strains under different saline conditions (C = control, SS = short-term salt stress, LS = long-term salt stress).

Figure 3 .
Figure 3. KEGG enrichment analysis of genes up-and down-expressed in ΔLeuRS (a,b) and WT (c,d), respectively (C = control, SS = short-term salt stress, LS = long-term salt stress).Figure 3. KEGG enrichment analysis of genes up-and down-expressed in ∆LeuRS (a,b) and WT (c,d), respectively (C = control, SS = short-term salt stress, LS = long-term salt stress).

Figure 3 .
Figure 3. KEGG enrichment analysis of genes up-and down-expressed in ΔLeuRS (a,b) and WT (c,d), respectively (C = control, SS = short-term salt stress, LS = long-term salt stress).Figure 3. KEGG enrichment analysis of genes up-and down-expressed in ∆LeuRS (a,b) and WT (c,d), respectively (C = control, SS = short-term salt stress, LS = long-term salt stress).

Figure 4 .
Figure 4. Heat map analysis of relative differential expression levels of some DEGs in compared groups of ΔLeuRS mutants and WT strains under distinct osmotic stress conditions.

Figure 4 .
Figure 4. Heat map analysis of relative differential expression levels of some DEGs in compared groups of ∆LeuRS mutants and WT strains under distinct osmotic stress conditions.

J 17 Figure 6 .
Figure 6.Conceptual profile showing enriched pathways of DEGs and metabolites between ΔLeuRS mutants and WT strains.DEGs involved in sexual and asexual processes (a).DEGs and metabolites were related to the metabolic pathways of amino acids, sugars, organic acids, and fatty acids (b).Genes marked with red or green color showed that these were up-and down-regulated at different time points.

Figure 6 .
Figure 6.Conceptual profile showing enriched pathways of DEGs and metabolites between ∆LeuRS mutants and WT strains.DEGs involved in sexual and asexual processes (a).DEGs and metabolites were related to the metabolic pathways of amino acids, sugars, organic acids, and fatty acids (b).Genes marked with red or green color showed that these were up-and down-regulated at different time points.