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Glutamine Synthetase Contributes to the Regulation of Growth, Conidiation, Sclerotia Development, and Resistance to Oxidative Stress in the Fungus Aspergillus flavus

The basic biological function of glutamine synthetase (Gs) is to catalyze the conversion of ammonium and glutamate to glutamine. This synthetase also performs other biological functions. However, the roles of Gs in fungi, especially in filamentous fungi, are not fully understood. Here, we found that conditional disruption of glutamine synthetase (AflGsA) gene expression in Aspergillus flavus by using a xylose promoter leads to a complete glutamine deficiency. Supplementation of glutamine could restore the nutritional deficiency caused by AflGsA expression deficiency. Additionally, by using the xylose promoter for the downregulation of AflgsA expression, we found that AflGsA regulates spore and sclerotic development by regulating the transcriptional levels of sporulation genes abaA and brlA and the sclerotic generation genes nsdC and nsdD, respectively. In addition, AflGsA was found to maintain the balance of reactive oxygen species (ROS) and to aid in resisting oxidative stress. AflGsA is also involved in the regulation of light signals through the production of glutamine. The results also showed that the recombinant AflGsA had glutamine synthetase activity in vitro and required the assistance of metal ions. The inhibitor molecule L-α-aminoadipic acid suppressed the activity of rAflGsA in vitro and disrupted the morphogenesis of spores, sclerotia, and colonies in A. flavus. These results provide a mechanistic link between nutrition metabolism and glutamine synthetase in A. flavus and suggest a strategy for the prevention of fungal infection.


Introduction
Aspergillus flavus (A. flavus) is a saprophytic, pathogenic, and conditional plant fungus that invades important crops such as peanuts and corn during storage and transportation [1][2][3]. A. flavus is not only a plant pathogenic fungus but also causes invasive aspergillosis, threatening human life [4,5]. In addition, this fungus produces toxic secondary metabolites (aflatoxins) that have been recognized as notorious carcinogenic natural contaminants since their discovery [6][7][8]. Aflatoxin B1 (AFB1) has strong carcinogenic, teratogenic, and genotoxic properties [9]. Understanding the mechanisms of development and toxin synthesis of A. flavus can greatly improve the control strategies for fungal contamination. Therefore, how to effectively prevent and inhibit the infestation of A. flavus has become a major challenge.

Identification of AflGsA in A. flavus
The sequence of the A. flavus Gs (AflGsA) protein was obtained from the NCBI gene database (AFLA_051930), which exhibited 63% similarity to GsA in Saccharomyces cerevisiae (S. cerevisiae). All of the analyzed proteins contained two conserved domains ( Figure 1A). The phylogenetic tree analysis based on AflGsA and other homologous proteins showed that AflGsA was highly conserved in Aspergillus spp. ( Figure 1B). The expression profiles of AflgsA were monitored by quantitative real-time PCR (qRT-PCR) at vegetative growth (VG), conidial development (CON), aflatoxin synthesis (AS), and sclerotial development (SD) stages [30]. The transcript level of AflgsA was the highest in the AS stage, whereas the lowest transcript level was reached in the SD stage ( Figure 1C). The differences in expression patterns suggest that AflgsA may perform different functions at different stages in A. flavus.

AflgsA Is an Essential Gene for the Growth of A. flavus
To investigate the function of AflgsA in A. flavus, we first tried to obtain the AflgsA deletion strain by homologous recombination but failed. A better alternative strategy is to construct a xylose promoter mutant strain ( xyIP AflgsA) for AflgsA functional verification ( Figure 2A). After confirmation by PCR ( Figure 2B) and DNA sequencing ( Figure S1), the mutant strain and WT strain were incubated in YXT medium (containing xylose) and YGT medium (without xylose) for four days at 37 • C in the dark. The mutant strain was completely unable to grow in the xylose-free medium, while growth was partially restored after the addition of xylose ( Figure 2C,D). Additionally, a gradient increase in the colony diameter, mycelial tip, and conidial gemination of xyIP AflgsA strain was found with an increasing concentration of xylose ( Figure S2). These results suggest that glutamine synthetase is essential for the growth of A. flavus. Expression patterns of AflgsA were tested by qRT-PCR at vegetative growth (VG), conidial development (CON), aflatoxin synthesis (AS), and sclerotial development (SD) stages. * indicates a significance level of p < 0.05, ** indicates a significance level of p < 0.01, and *** indicates a significance level of p < 0.001 based on one-way ANOVA with three biological replicates.

AflgsA Is an Essential Gene for the Growth of A. flavus
To investigate the function of AflgsA in A. flavus, we first tried to obtain the AflgsA deletion strain by homologous recombination but failed. A better alternative strategy is to construct a xylose promoter mutant strain ( xyIP AflgsA) for AflgsA functional verification ( Figure 2A). After confirmation by PCR ( Figure 2B) and DNA sequencing ( Figure S1), the Expression patterns of AflgsA were tested by qRT-PCR at vegetative growth (VG), conidial development (CON), aflatoxin synthesis (AS), and sclerotial development (SD) stages. * indicates a significance level of p < 0.05, ** indicates a significance level of p < 0.01, and *** indicates a significance level of p < 0.001 based on one-way ANOVA with three biological replicates. completely unable to grow in the xylose-free medium, while growth was partially restored after the addition of xylose ( Figure 2C,D). Additionally, a gradient increase in the colony diameter, mycelial tip, and conidial gemination of xyIP AflgsA strain was found with an increasing concentration of xylose ( Figure S2). These results suggest that glutamine synthetase is essential for the growth of A. flavus.

Glutamine Restores the Growth Defect of the xyIP AflgsA Strain
We found that the addition of glutamine (GluN) to the xylose-free or xylose medium restored the growth of the xyIP AflgsA strain ( Figures 3A,B and S3). Meanwhile, the ammonium tartrate (NH4 + ) or glutamate (Glu) supplement was unable to promote colony growth ( Figures 3A,B and S4). This suggested that the growth defect of the mutant strain was caused by the lack of glutamine synthetase. However, the xyIP AflgsA strain showed different colony color compared to the WT strain after GluN supplementation ( Figure 3A). Moreover, the growth of the xyIP AflgsA strain was also inhibited by adding both GluN and NH4 + ( Figure 3B,C). This phenomenon may be due to competition or inhibition of GluN uptake by excess NH4 + [21]. The mechanisms concerning the type of nitrogen sources that mediate the absorption pathway by AflgsA in the cell need to be further explored.

Glutamine Restores the Growth Defect of the xyIP AflgsA Strain
We found that the addition of glutamine (GluN) to the xylose-free or xylose medium restored the growth of the xyIP AflgsA strain ( Figures 3A,B and S3). Meanwhile, the ammonium tartrate (NH 4 + ) or glutamate (Glu) supplement was unable to promote colony growth ( Figures 3A,B and S4). This suggested that the growth defect of the mutant strain was caused by the lack of glutamine synthetase. However, the xyIP AflgsA strain showed different colony color compared to the WT strain after GluN supplementation ( Figure 3A). Moreover, the growth of the xyIP AflgsA strain was also inhibited by adding both GluN and NH 4 + ( Figure 3B,C). This phenomenon may be due to competition or inhibition of GluN uptake by excess NH 4 + [21]. The mechanisms concerning the type of nitrogen sources that mediate the absorption pathway by AflgsA in the cell need to be further explored.

AflGsA Is Important for Conidia Development of A. flavus
To further determine the effect of AflgsA on A. flavus, we monitored the transcript level of AflgsA in the xyIP AflgsA strain and found that it was less than half that in the WT ( Figure 4A). In addition, the xyIP AflgsA strain exhibited smaller spore heads of the child seat and sparser peduncles by microscopic observation ( Figure 4B). Statistical analysis also showed a significant reduction in the number of spores in the xyIP AflgsA strain compared to that in the WT strain ( Figure 4C). Further analysis showed that the transcript levels of both regulatory genes for conidia, brlA, and abaA, were reduced by about half in the xyIP AflgsA strain compared to that in WT ( Figure 4D). These results suggest that AflgsA may be involved in the upstream regulation of abaA and brlA genes during spore development of A. flavus, and this further regulates the production of conidia.

AflGsA Is Important for Conidia Development of A. flavus
To further determine the effect of AflgsA on A. flavus, we monitored the transcript level of AflgsA in the xyIP AflgsA strain and found that it was less than half that in the WT ( Figure 4A). In addition, the xyIP AflgsA strain exhibited smaller spore heads of the child seat and sparser peduncles by microscopic observation ( Figure 4B). Statistical analysis also showed a significant reduction in the number of spores in the xyIP AflgsA strain compared to that in the WT strain ( Figure 4C). Further analysis showed that the transcript levels of both regulatory genes for conidia, brlA, and abaA, were reduced by about half in the xyIP AflgsA strain compared to that in WT ( Figure 4D). These results suggest that AflgsA may be involved in the upstream regulation of abaA and brlA genes during spore development of A. flavus, and this further regulates the production of conidia. (C) The number of conidia produced by the above two A. flavus strains. ND indicates no detection. *** indicates a significance level of p < 0.001 based on t-tests with three replicates.

AflGsA Contributes to the Production of Sclerotia but Not to Toxin Synthesis in A. flavus
Sclerotia are important reproductive structures of Aspergillus flavus, and they aid in survival under harsh conditions. The sclerotia produced by the xyIP AflgsA strain were significantly fewer compared to those in the WT strain ( Figure 5A,B). In addition, the transcript levels of the sclerotia-related genes nsdC and nsdD were significantly lower in the xyIP AflgsA strain compared to those in the WT strain, only about half of those in the WT strain ( Figure 5C). In addition, the sclerotium production defect in the xyIP AflgsA strain could not be recovered by the addition of a GluN supplement ( Figure S5). These results suggest that AflGsA is important for sclerotia production, and its coding gene may influence sclerotia production by being involved in the upstream regulation of nsdC and nsdD. We also investigated the production of the toxic secondary metabolite AFB1 in A. flavus and found no significant differences in toxin production between the xyIP AflgsA strain and the WT ( Figure 5D,E). This suggests that AflgsA is not involved in the regulation of aflatoxin production.

AflGsA Balances ROS and Resists Oxidative Stress in A. flavus
A high concentration of ROS causes oxidative damage, disrupting cell metabolism and causing apoptosis. It was found that more ROS were produced in the low-expression state of AflgsA ( Figure 6A). When adding hydrogen peroxide to simulate the oxidative stress state, the inhibition rate of the xyIP AflgsA strain was significantly higher than that of the WT ( Figure 6B,C), but this situation was significantly restored by the addition of GluN ( Figure 6B,C). A similar phenotype was also shown in the addition of tBOOH to simulate the oxidative stress state ( Figure S6). Further study revealed that there was no significant change in the transcript level of superoxide dismutase gene sod. However, the transcript level of the catalase gene cat was significantly lower in the xyIP AflgsA strain compared to that in the WT strain ( Figure 6D). These results suggest that GluN, the catalytic product of AflGsA, plays an important role in resistance to oxidative stress.

AflGsA Contributes to the Production of Sclerotia but Not to Toxin Synthesis in A. flavus
Sclerotia are important reproductive structures of Aspergillus flavus, and they aid in survival under harsh conditions. The sclerotia produced by the xyIP AflgsA strain were significantly fewer compared to those in the WT strain ( Figure 5A,B). In addition, the transcript levels of the sclerotia-related genes nsdC and nsdD were significantly lower in the xyIP AflgsA strain compared to those in the WT strain, only about half of those in the WT strain ( Figure 5C). In addition, the sclerotium production defect in the xyIP AflgsA strain could not be recovered by the addition of a GluN supplement ( Figure S5). These results suggest that AflGsA is important for sclerotia production, and its coding gene may influence sclerotia production by being involved in the upstream regulation of nsdC and nsdD. We also investigated the production of the toxic secondary metabolite AFB1 in A. flavus and found no significant differences in toxin production between the xyIP AflgsA strain and the WT ( Figure 5D,E). This suggests that AflgsA is not involved in the regulation of aflatoxin production.

AflGsA Is Involved in Light Signaling Pathways in A. flavus
The colony diameter of A. flavus at 37 • C in the light was significantly smaller than that in the dark ( Figure 7A). We also found that the xyIP AflgsA strain exhibited significantly higher inhibition rates under light relative to those of the WT strain ( Figure 7A,B), but this inhibition of growth was restored with the addition of glutamine ( Figure 7A,B). Further studies found that A. flavus produced more ROS under light ( Figure 7C), indicating that AflGsA could regulate ROS production under light conditions.

Recombinant AflGsA (rAflGsA) Has Glutamine Synthetase Activity
The recombinant protein rAflGsA (rAflGsA with 6× His tag, 6× His-rAflGsA) was successfully expressed and then purified by Ni-NTA column chromatography ( Figure 8A). Recombinant protein activity was measured using the classical Gs enzymatic activity assay (see Section 5), and the results showed that rAflGsA has glutamine synthetase activity ( Figure 8B). We further analyzed whether divalent metal ions affected the enzyme activity of rAflGsA. Based on the results of activity assays, the highest activity was observed with Mg 2+ ions (control), while the activities with Mn 2+ and Ca 2+ ions were 81% and 72%, respectively ( Figure 8C). Glutamine synthetase activity with the addition of Cu 2+ ions was only 18% ( Figure 8C). From the results above, it appears that the activity of rAflGsA requires the assistance of specific metal ions. 2.9. L-α-Aminoadipic Acid Is a Potential Inhibitor for rAflGsA and A. flavus L-α-aminoadipic acid is a specific gliotoxin in vitro, and this chemical was reported to inhibit Gs activity in rats [31]. In this study, we found that the inhibitor L-α-aminoadipic acid suppressed the activity of rAflGsA with an IC 50 value of 288.1 µM in vitro ( Figure 8D). The results also showed that L-α-aminoadipic acid prevented the growth and conidial gemination of A. flavus with increasing concentrations (Figures 9A,B and S7). The morphology of conidia was influenced by an increasing concentration, and the spore heads became smaller ( Figure 9C). The statistics revealed a gradient decrease in the number of spores of A. flavus with an increasing concentration of L-α-aminoadipic acid ( Figure 9D). When observing the effect of L-α-aminoadipic acid on the production of sclerotia, we found a significant decrease in the number of sclerotia when 1.6 mg/mL of L-α-aminoadipic acid was added ( Figure 9E,F). In contrast, the amount of aflatoxin did not change with an increasing concentration of added L-α-aminoadipic acid ( Figure 9G,H). In view of the results above, it is clear that L-α-aminoadipic acid is an effective inhibitor of rAflGsA and consequently for A. flavus. Therefore, we speculate that AflGsA in A. flavus is an ideal candidate target for the L-α-aminoadipic acid inhibitor.

AflGsA Balances ROS and Resists Oxidative Stress in A. flavus
A high concentration of ROS causes oxidative damage, disrupting cell metabolism and causing apoptosis. It was found that more ROS were produced in the low-expression state of AflgsA ( Figure 6A). When adding hydrogen peroxide to simulate the oxidative stress state, the inhibition rate of the xyIP AflgsA strain was significantly higher than that of the WT (Figure 6B,C), but this situation was significantly restored by the addition of GluN

AflGsA Is Involved in Light Signaling Pathways in A. flavus
The colony diameter of A. flavus at 37 °C in the light was significantly smaller than that in the dark ( Figure 7A). We also found that the xyIP AflgsA strain exhibited significantly higher inhibition rates under light relative to those of the WT strain ( Figure 7A,B), but this inhibition of growth was restored with the addition of glutamine ( Figure 7A,B). Further

Recombinant AflGsA (rAflGsA) Has Glutamine Synthetase Activity
The recombinant protein rAflGsA (rAflGsA with 6× His tag, 6× His-rAflGsA) was successfully expressed and then purified by Ni-NTA column chromatography ( Figure  8A). Recombinant protein activity was measured using the classical Gs enzymatic activity assay (see Section 5), and the results showed that rAflGsA has glutamine synthetase activity ( Figure 8B). We further analyzed whether divalent metal ions affected the enzyme activity of rAflGsA. Based on the results of activity assays, the highest activity was observed with Mg 2+ ions (control), while the activities with Mn 2+ and Ca 2+ ions were 81% and 72%, respectively ( Figure 8C). Glutamine synthetase activity with the addition of Cu 2+ ions was only 18% ( Figure 8C). From the results above, it appears that the activity of rAflGsA requires the assistance of specific metal ions.

L-α-Aminoadipic Acid Is a Potential Inhibitor for rAflGsA and A. flavus
L-α-aminoadipic acid is a specific gliotoxin in vitro, and this chemical was reported to inhibit Gs activity in rats [31]. In this study, we found that the inhibitor L-α-aminoadipic acid suppressed the activity of rAflGsA with an IC50 value of 288.1 μM in vitro ( Figure  8D). The results also showed that L-α-aminoadipic acid prevented the growth and conidial gemination of A. flavus with increasing concentrations (Figures 9A,B and S7). The morphology of conidia was influenced by an increasing concentration, and the spore heads became smaller ( Figure 9C). The statistics revealed a gradient decrease in the number of

Discussion
Glutamine synthetase is responsible for catalyzing the conversion of glutamine from ammonium and glutamate as well as being the central enzyme for nitrogen assimilation [32]. Unlike Rhizobium meliloti (R. meliloti) [33] and Magnaporthe oryzae (M. oryzae) [24], which have three Gs proteins, we identified only one putative glutamine synthetase (AflGsA) in A. flavus. Furthermore, a previous report on R. meliloti stated that only simultaneous knockdown of all three Gs genes could cause a complete glutamine nutritional defect [33]. In A. flavus, only one protein has glutamine synthetase activity, and there is no alternative pathway for glutamine synthesis. To study the biofunction of glutamine synthetase in A. flavus, we constructed the xyIP AflgsA mutant strain that has a complete glutamine nutrientdeficient mutation in the YGT medium.
In A. flavus, the addition of glutamine to the YGT medium partially restored the growth defect of the xyIP AflgsA strain. However, in contrast to the pigmented colonies with conidia produced in A. nidulans [21], the addition of glutamine to the A. flavus mutant strain resulted in the formation of pigmentation defects similar to M. oryzae and G. fujikuroi [15,24]. This suggests that the glutamine synthetase regulated the production of A. flavus pigments, unlike that in A. nidulans. In addition, the phenotype after glutamine addition was inhibited by the addition of NH 4 + , which may be due to the competitive inhibition of GluN by NH 4 + [21]. The transcript level of ghd gene encoding glutamate dehydrogenase was elevated in the xyIP AflgsA strain compared to that in WT ( Figure S8A). Glutamate dehydrogenase also plays an important role in ammonium assimilation. This may be a balancing mechanism for nitrogen regulation in A. flavus.
Like many pathogenic fungi, conidia production and sclerotia formation are important steps in the life cycle of A. flavus. It was found that the AflgsA of A. flavus plays an important role in the production of conidia. Further results also showed that AflgsA regulates spore production by regulating the transcript levels of the regulatory genes brlA and abaA for the production of conidia. Similarly to ∆Mogln2 in M. oryzae [24], the inhibition of AflgsA function in A. flavus was followed by a reduction in the number of conidial peduncles and a smaller head of child seats compared to those in the WT, which may be a reason for the reduction of A. flavus spores. In addition, AflgsA in A. flavus regulates sclerotia formation by affecting the sclerotia-production-related genes nsdC and nsdD. All these results suggest that the sophisticated role of AflgsA in multiple developmental stages of A. flavus is related to its glutaminyl transferase activity. Glutamine synthetase in G. fujikuroi affects the synthesis of gibberellin (GA) and bikaverin metabolites [15]. In contrast, our study showed that both the xyIP AflgsA mutant and the inhibitor-treated strains produced aflatoxin normally, revealing that AflgsA is not involved in aflatoxin synthesis in A. flavus.
ROS is an unavoidable and harmful by-product of oxidative metabolism, and ROS dynamic balance is essential for the development of the fungus [34,35]. The inhibition of glutamine synthetase or light irradiance led to more ROS production and had an inhibitory feedback effect on growth in A. flavus that was restored by the addition of glutamine. Photorespiration in plants leads to high production of ROS [36], and inhibition of Gs in A. palmeri leads to impaired function of photorespiration accompanied by cell apoptosis [17,22]. Therefore, we hypothesized that glutamine synthetase regulated ROS-mediated inhibition of the growth of A. flavus under light conditions. Furthermore, glutamine synthetase can regulate the metabolism in relation to oxidative stress in cyanobacteria [37]. In M. oryzae, the ∆Mogln2 strain also results in a high sensitivity to H 2 O 2 [24]. In this study, the AflgsA mutant strain of A. flavus was highly sensitive to oxidative stress, and this sensitivity was restored by supplementing glutamine. Further studies showed that the transcript level of catalase was significantly reduced in the AflgsA mutant strain, suggesting that the glutamine synthetase pathway resists oxidative stress by regulating the transcriptional level of catalase.
We obtained the rAflGsA protein from E. coli with a purity of up to 95% and confirmed that it had glutamate synthase activity. Reports show that glutamine synthetase has positive cooperativity with different cofactors and metal ions [38,39]. Our further study clarified that the glutamine synthesis activity of rAflGsA is dependent on divalent metal ions and that the highest enzyme activity is achieved by the addition of Mg 2+ and Mn 2+ ions. This finding was consistent with the results of cation preference towards recombinant glutamine synthetase from Psychrotrophic Bacterium [40] and Mangrove [41].
L-α-aminoadipic acid is a specific gliotoxin in vitro [42] and is a neuroexcitatory metabolite that reduces extracellular kynurenic acid levels in a dose-dependent manner [43]. However, it is not mentioned in other studies whether L-α-aminoadipic acid has antifungal activity. Our study showed that L-α-aminoadipic acid is an inhibitor for rAflGsA in vitro, and that it also prevents the growth, spore production, and sclerotia formation of A. flavus in vivo. In agreement with the phenotype of the xyIP AflgsA strain, the addition of L-αaminoadipic acid had no effect on aflatoxin synthesis. In addition, lower concentrations of L-α-aminoadipic acid (0.8 mg/mL and 1.6 mg/mL) did not affect the transcript level of AflgsA, but a higher dosage (3.2 mg/mL) seemed to decrease its transcriptional ( Figure S8B). This suggests that L-α-aminoadipic acid may also have other unknown targets in A. flavus. Overall, these results suggest that L-α-aminoadipic acid is a potential inhibitor of A. flavus, and thus AflGsA may be an ideal target for L-α-aminoadipic acid in A. flavus. The study of the inhibition mechanism of L-α-aminoadipic acid against A. flavus is important for the prevention of A. flavus. This may be a very meaningful research direction in the future.

Conclusions
At present, many studies have attempted to elucidate the function of glutamine synthetase in organisms. However, the understanding of the biofunctional diversity of glutamine synthetase in filamentous fungi, especially in A. flavus, seems to have been relatively neglected. In this study, we found that AflGsA performed its activity as a glutamine synthetase and that it played a divergent role in the conidia production and sclerotia formation in A. flavus. It has an important role in the homeostasis of ROS and resistance to oxidative stress in A. flavus. In addition, L-α-aminoadipic acid inhibited both rAflGsA and A. flavus and thus was considered as a potential antifungal candidate for further study.

Strains and Culture Conditions
E. coli DH5a and BL21 (DE3) were used for plasmid DNA preparation and expression of the recombinant AflGsA (rAflGsA) protein, respectively. A. flavus WT and xyIP AflgsA strains were cultured in YGT (5 g/L yeast extract, 20 g/L glucose, and 1 mL/L trace elements) and YXT (5 g/L yeast extract, 20 g/L xylose, and 1 mL/L trace elements) media at 37 • C in the dark. Then, 1.5% agar was added to obtain solid media. To study aflatoxin production, YXT medium containing 1 g/L MgSO 4 ·7H 2 O was used at 29 • C.

Sequence Analysis
The NCBI database was used to search for the AflgsA sequence of A. flavus (AFLA_051930). The homologous protein sequence of AflGsA from A. flavus was retrieved by BLAST. The retrieved homologous protein sequences were analyzed by domain analysis using Uniprot Tools and mapped using DOG 2.0 software. The MAGE 7.0 software was used for multiple sequence alignment of the protein sequences, and the maximum likelihood method was used to construct the phylogenetic tree.

Construction and Identification of Mutant Strain
All of the primers used in this study were shown in Table S1. To obtain the xyIP AflgsA mutant strain, we followed the method previously described in the literature [44]. An AflgsA xylose promoter mutant cassette was fused by overlapping extension PCR (gsA-xolap-F and gsA-xolap-R primers were used) to an upstream fragment of AflgsA, a marker gene (A. fumigatus pyrG), the xyIP xylose conditional promoter [45], and the AflgsA CDS fragment. A. flavus CA14 was used as a starting strain during the preparation of protoplasts [46]. The AflgsA gene's promoter was converted to a xylose promoter by homologous recombination [44]. The gsA-A-F and gsA-CDS-R primers were used in the identification of xyIP AflgsA transformants by PCR. The AflgsA-F and AflgsA-R primers were used to identify the sequence by qRT-PCR.

Analysis of the Growth, Conidial Production, and Sclerotia Formation of A. flavus
For colony diameter evaluation, YGT and YXT containing 10 mM glutamine (GluN) or ammonium tartrate (NH 4 + ) were points inoculated with 10 6 conidia and incubated in the dark at 37 • C for four days. Spores were eluted with 2 mL of spore eluate and counted under a microscope using a hemocytometer plate to determine the number of spores after incubation in the dark at 37 • C for four days [44]. To observe conidiophore formation, 10 6 conidia were incubated in YXT medium at 37 • C in the dark for two days, and the surface mycelium was scraped off. The colonies were cut out and placed on cover breaks and incubated for 12 h at 37 • C, then observed using a microscope. For sclerotia production analysis, 10 6 conidia were incubated in YXT medium at 37 • C in the dark for seven days, and the morphology of sclerotia was recorded by rinsing off the mycelium with 75% ethanol [47]. For aflatoxin extraction, 10 6 conidia were incubated in YXT containing 1 g/L MgSO 4 ·7H 2 O at 29 • C in the dark for five days. The toxin in the medium was extracted using chloroform and detected using TLC [44].

Detection of ROS in A. flavus
To detect the ROS in A. flavus, WT and xyIP AflgsA strains were incubated in YXT liquid medium at 37 • C for 24 h. Mycelia were washed three times with a phosphatebuffered saline (PBS) buffer. The collected mycelia were incubated with a 10 µM DCFH-DA fluorescent probe (Beyotime, Nantong, China) for 30 min at 37 • C. After washing three times with PBS, the mycelium was placed on a slide, and ROS content was identified by microscopy. Fluorescence emission of DCFH-DA was excited at 488 nm.

Quantitative Real-Time PCR
The mycelia of the strains of A. flavus were collected after 48 h of incubation. The collected mycelia were ground in liquid nitrogen, and total RNA was extracted with the TRIzol (Biomarker Technologies, Beijing, China) reagent. RNA was translated into cDNA using a reverse transcription kit (Thermo Scientific, Waltham, MA, USA). The cDNA was then used as a template for quantitative PCR with specific primers [48].

Purification of Recombinant rAflGsA Protein and Determination of Enzyme Activity
The cDNA of AflgsA from A. flavus was expanded and cloned into the pET-28a expression vector. The expressed recombinant AflGsA (rAflGsA) protein was purified using Ni-NTA column chromatography [44]. The activity of rAflGsA was then measured at 540 nm using the classical glutamine synthetase enzyme activity assay [40]. Different divalent metal ions were added to assay the enzyme activity with a final concentration of 20 mM. Enzyme activity was measured after reacting at 37 • C for one hour.

Statistical Analysis
GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) was used for data statistics and analysis. All of the analyses had at least three biological replicates if not specifically indicated.

Supplementary Materials:
The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/toxins14120822/s1, Table S1: Oligonucleotide primers used in this study; Figure S1: Genome sequencing validation of the xyIP AflgsA strain of A. flavus. AflgsA AP is the upstream noncoding region of the AflgsA gene. The mutations in the AflgsA CDS are synonymous mutations; Figure S2: Analysis of the growth, mycelial morphology, conidial germination, and conidiophore formation of WT and xyIP AflgsA strains. (G) The number of conidia produced by the above two A. flavus strains. ND indicates no detection. ** indicates a significance level of p < 0.01, and *** indicates a significance level of p < 0.001 based on t-tests with three replicates. The medium containing 10 g/L xylose: 5 g/L yeast extract, 10 g/L glucose, 10 g/L xylose, 1 mL/L trace elements, and 1.5% agar. The medium containing 20 g/L xylose (YXT medium): 5 g/L yeast extract, 20 g/L xylose, 1 mL/L trace elements, and 1.5% agar; Figure S3  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.