MaAreB, a GATA Transcription Factor, Is Involved in Nitrogen Source Utilization, Stress Tolerances and Virulence in Metarhizium acridum

The nitrogen catabolite repression (NCR) pathway is involved in nitrogen utilization, in which the global GATA transcription factor AreA plays an indispensable role and has been reported in many fungi. However, relatively few studies are focused on AreB, another GATA transcription factor in the NCR pathway and the functions of AreB are largely unknown in entomopathogenic fungi. Here, we characterized MaAreB in the model entomopathogenic fungus Metarhizium acridum. Sequence arrangement found that MaAreB had a conserved GATA zinc finger DNA binding domain and a leucine zipper domain. Disruption of MaAreB affected the nitrogen utilization and led to decelerated conidial germination and hyphal growth, decreased conidial yield, and lower tolerances to UV-B irradiation and heat-shock. Furthermore, the MaAreB mutant (ΔMaAreB) exhibited increased sensitivity to CFW (Calcofluor white), decreased cell wall contents (chitin and β-1,3-glucan) and reduced expression levels of some genes related to cell wall integrity, indicating that disruption of MaAreB affected the cell wall integrity. Bioassays showed that the virulence of the ΔMaAreB strain was decreased in topical inoculation but not in intra-hemocoel injection. Consistently, deletion of MaAreB severely impaired the appressorium formation and reduced the turgor pressure of appressorium. These results revealed that MaAreB regulated fungal nitrogen utilization, cell wall integrity and biological control potential, which would contribute to the functional characterization of AreB homologous proteins in other insect fungal pathogens, and even filamentous fungi.


Introduction
There are many fungi in the natural environment; some are pathogenic and are a threat to human health, agriculture and forestry, such as Aspergillus fumigatus, Candida albicans, A. flavus, Fusarium graminearum, and Magnaporthe oryzae [1,2]. Some are important biological control fungi, which can be used to control crop diseases and pests, such as Trichoderma harzianum, Beauveria bassiana and Metarhizium anisopliae [3][4][5]. Entomopathogenic fungi, as an important class of insecticidal microbes, are important regulators of the insect population in nature, and have been applied to the control of agricultural and forestry pests [6,7]. Similar to many plant fungal pathogens, the conidia of insect fungal pathogens, like B. bassiana, could adhere to the host cuticles and penetrate these via the infection structure appressorium under the combination of turgor pressure and cuticle-degrading enzymes [8][9][10]. Under natural conditions, fungi have to cope with a variety of environmental challenges, such as temperature, ultraviolet rays, nutritional restriction conditions, etc. [11].
Nitrogen is a necessary influencing factor for fungal survival. Fungi can uptake some kinds of compound as nitrogen sources, but other nitrogen sources will not be assimilated predicted online via SMART interface (http://smart.emblheidelberg.de/) (accessed on 3 March 2021). Multiple sequence alignments were performed by the DNAMAN program, and the neighbor-joining tree was constructed by MEGA6 under the condition of default settings and 1000 bootstrap replicates [29].

Construction of the Mutants
To construct the MaAreB-disruption vectors, about 1000-bp up-and down-stream fragments of MaAreB were cloned from WT genomic DNA and inserted into pK2-SM-F and pK2-SM-R vectors [30] to form the gene knockout vectors, pK2-SM-MaAreB-F and pK2-SM-MaAreB-R ( Figure S1A). The DNA and promoter sequence of MaAreB was amplified and inserted into pK2-sur vector [31] to form the complementation plasmid, pK2-sur-MaAreB ( Figure S1B). Both knockout and complementation recombinant vectors were transferred into AGL1 for fungal transformation [32]. The ∆MaAreB and CP transformants were screened on CZA plates supplemented with 0.5 g/L glufosinate-ammonium (Sigma, St. Louis, MO, USA) or 0.02 g/L chlorimuron ethyl (Sigma, Bellefonte, PA, USA), respectively. Firstly, the fragments of target gene and resistance genes were amplified for preliminary verification of the transformants. On the other hand, Southern blotting was used for further verification via DIG High Prime DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland) ( Figure S1C). All primers are listed in Table S1.

Growth Characteristic Assays
The conidia of WT, ∆MaAreB and CP strains, cultured on 1/4SDAY for 15 days, were used for preparation of conidial suspensions (10 6 or 10 7 conidia/mL). For examination of the conidial germination rate and observation of hyphal morphological development, 100 µL conidial suspensions (10 7 conidia/mL) were pipetted and evenly coated on 1/4SDAY plate, followed by incubating for hours, then the germination of conidia was counted every two hours [33], and photographs taken every three hours, to record the mycelial growth, with a digital light microscope (MOTIC, Xiamen, China). For determination of the conidial yield, 2 µL conidial suspensions (10 6 conidia/mL) were spotted onto 1/4SDAY or modified CZA medium and cultured at 28 • C for days, and every colony was broken in 1 mL 0.1% Tween-80 to count the number of conidia with a hemocytometer on different days [33]. To analyze the nitrogen utilization, we improved the CZA medium and replaced nitrate with 10 mM (NH 4 ) 2 SO 4 , Glutamine, Glutamate or Proline to act as the sole nitrogen source, respectively. Two microliters of conidial suspensions (10 6 conidia/mL) were spotted onto modified CZA medium and cultured at 28 • C for 7 days to observe the colony morphology and determine the conidial yield.

Stress Tolerance Assays
The tolerances of M. acridum to UV-B and heat-shock resistance were tested as described previously [34]. Briefly, in the UV-B tolerance test, 50 µL conidial suspensions (10 7 conidia/mL) of different fungal strains were evenly spread on 1/4SDAY plates, followed by exposure to UV-B irradiation (1350 MW/m 2 ) for 1.25 h, 2.50 h, 3.75 h and 5.00 h, which provided total doses of 6.075, 12.150, 18.225, 24.300 kJ/m 2 , respectively. A 40-W fluorescent lamp was used to provide the UV-B irradiation, and the solid plates were evenly distributed side by side on both sides under the lamp. For heat-shock tolerance test, the conidial suspensions (10 7 conidia/mL) of each strain were treatment in a 45 • C water bath for hours, followed by spreading 50 µL evenly on 1/4SDAY every three hours. All strains treated with UV-B irradiation and heat-shock were cultured for 20 h to examine the conidial germination.
To analyze cell wall integrity, 2 µL conidial suspensions (10 6 conidia/mL) of each strain were spotted on 1/4SDAY (control) or supplemented with 150 µg/mL cell wall perturbing agent CFW, then incubated for 7 days at 28 • C, followed by observation of the colony growth and calculation of the relative inhibition rates [35].

Determination of Cell Wall Composition
The cell wall contents, chitin, β-1,3-glucan and mannoproteins, of M. acridum were determined according to the previous study [36]. Briefly, hyphae of the WT, ∆MaAreB and CP strains were respectively inoculated in a 40 mL 1/4SDY liquid medium (1/4 SDAY without agar) and cultured at 28 • C with 220 rpm for 3 days. Then, the hyphae were harvested and washed with 2% SDS four times, followed by lyophilizing for 24 h. Five milligrams (dry weight) hyphae were consumed for each determination. For determination of the chitin content, hydrochloric acid was used to hydrolyze fungal cell wall, and glucosamine was used as the standard. For determination of β-1,3-glucan content, sodium hydroxide was used to treat the fungal cell wall to obtain the alkali-insoluble fraction, followed by degrading with zymolyase 100-T, and laminaripentaose (Sigma, St. Louis, MO, USA) was used as the standard. To detect the content of mannoprotein, sodium hydroxide was used to extract the mannoprotein in each strain. The standard curve of bovine serum albumin was made via Folin-phenol reagent method.

Quantitative Reverse Transcription PCR (qRT-PCR) Analysis
The mature (15-day-old) conidia on 1/4SDAY medium were harvested and used to detect the expression of genes involved in the tolerances to UV-B, heat-shock, or the cell wall integrity. Appressoria of each strain were collected from the locust hind wings after 27 h of incubation at 28 • C to detect the expression of genes involved in fungal adhesion, cuticle penetration, or glycerol synthesis. The RNAs of fungal cells (conidia and appressoria) were extracted with fungal RNA Kit (CoWin Biosciences, Beijing, China). PrimeScript TM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) was used for synthesising cDNA. qRT-PCR was performed using SYBR ® Premix Ex TaqTM (TaKaRa, Dalian, China). M. acridum gpdh (EFY84384) acted as the reference gene, and the data were analyzed according to the 2 −∆∆Ct method [37]. Forty cycles were performed for all experiments. The details of CT value, amplicon length, and PCR efficiencies for each primer pair are provided in supplementary Table S2. The primers used for qRT-PCR are listed in supplementary Table S1.

Insect Bioassays
Topical inoculation and intra-hemocoel injection methods were used to investigate the virulence of the WT, ∆MaAreB and CP strains as described previously [38]. Briefly, 5 µL 10 7 conidia/mL conidial suspensions were dropped on the pro-nota of a fifth-instar locust nymph as topical inoculation test, and liquid paraffin acted as control. Five microliters 10 6 conidia/mL of conidial suspensions were injected into the hemolymph of a fifth-instar locust nymph as intra-hemocoel injection test, and sterile water was used as the control. The bioassay environment was maintained in a stable photoperiod (16-h light and 8-h dark) with an ambient temperature of 28 • C. Three replicates of each treatment (n = 30 locusts) were performed, and the experiment was repeated three times. The mortality was recorded every half day until all the test insects had died. Virulence of each strain was estimated with the LT 50 (the mean 50% lethality time). In order to observe the growth of M. acridum in locust hemolymph, 600 µL blood for each treatment were collected to count the number of hyphal bodies and quantify the gDNA concentrations of the M. acridum, using quantitative PCR with the specificity ITS primers [39].

Infection Structure Development Assays
Conidial germinations and appressorium formations of the WT, ∆MaAreB and CP strains were determined as previously described [40]. Appressoria from different fungal strains were treated with different concentrations of PEG8000 to analyze the appressorial turgor pressure. The neutral lipids were stained with Nile Red as described previously [41], and the fluorescence intensity of the neutral lipids in appressorium was measured by ImageJ software.

Statistical Analysis
The data were presented as the mean ± SD and analyzed using ANOVA (one-way analysis of variance) with the SPSS 24.0 program. The differences were analyzed with Tukey's HSD test. Three biological replicates were performed for each result.

Bioinformatics Analysis and Generation of MaAreB Mutants
MaAreB was cloned from the genomic DNA of the WT strain; the DNA sequence was 1152 bp with one intron. The ORF (open reading frame) was 1098 bp, encoding 365 amino acids. The physical and chemical properties of MaAreB were analyzed in silico, showing that the protein mass was 39.77 kDa, and the isoelectric point was 4.76. MaAreB encoded a GATA transcription factor with a zinc finger DNA binding domain at the N-terminal and a leucine zipper domain at the C-terminal. Both domains were highly conserved in fungi, and the identity was 51.30% by protein sequence alignment of MaAreB with its homologs ( Figure 1A). The homologous proteins of AreB, such as Asd4, Gzf3, or Dal80, were found in many fungi, and the phylogenetic tree analysis showed that MaAreB was relatively close to the homologous proteins of filamentous fungi in evolution, and far from yeast ( Figure 1B).
According to the principle of homologous recombination, the ∆MaAreB strain was obtained by replacing the 1.1 kb MaAreB gene with the bar cassette ( Figure S1A). The CP strain was constructed by the principle of random insertion ( Figure S1B). All the fungal strains were verified by Southern blotting ( Figure S1C).

MaAreB Was Involved in the Regulation of Nitrogen Utilization
AreB is a core member of NCR pathway and plays an important role in nitrogen utilization [14]. Thus, the WT, ΔMaAreB and CP strains were grown on modified CZA media, which were replaced the nitrate in CZA with 10 mM (NH4)2SO4, glutamine, glutamate or proline, respectively. The results showed that the colonies of ∆MaAreB grown on CZA media containing various nitrogen resources, except for ammonium, were smaller compared to the WT or CP strain (Figure 2A, B). The conidial yield of the ∆MaAreB strain decreased slightly with no significant difference to the WT or CP strain

MaAreB Was Involved in the Regulation of Nitrogen Utilization
AreB is a core member of NCR pathway and plays an important role in nitrogen utilization [14]. Thus, the WT, ∆MaAreB and CP strains were grown on modified CZA media, which were replaced the nitrate in CZA with 10 mM (NH 4 ) 2 SO 4 , glutamine, glutamate or proline, respectively. The results showed that the colonies of ∆MaAreB grown on CZA media containing various nitrogen resources, except for ammonium, were smaller compared to the WT or CP strain (Figure 2A,B). The conidial yield of the ∆MaAreB strain decreased slightly with no significant difference to the WT or CP strain grown on glutamate as the sole nitrogen source, but was significantly decreased when nitrate, glutamine or proline was used as the sole nitrogen source ( Figure 2C). These data evidenced that MaAreB was involved in regulating nitrogen utilization. grown on glutamate as the sole nitrogen source, but was significantly decreased when nitrate, glutamine or proline was used as the sole nitrogen source ( Figure 2C). These data evidenced that MaAreB was involved in regulating nitrogen utilization.

Deletion of MaAreB Affected Conidial Germination, Hyphal Growth and Conidiation
To analyze the growth characteristics of the WT, ∆MaAreB and CP strains, we tested the conidial germinations, the macroscopic and microscopic growth morphologies, and the conidial yield of fungal strains grown on 1/4SDAY. The results showed that although there was no significant difference in colony morphology among the WT, ∆MaAreB and CP strains ( Figure 3A), the germination rate of ∆MaAreB strain was significantly slower at 2, 4, 6, 8 and 10 h ( Figure 3B). The GT50 (the mean 50% germination time) of the ∆MaAreB strain (6.90 ± 0.23 h) was significantly prolonged compared with the WT (5.25 ± 0.20 h) or CP (5.43 ± 0.19 h) strain ( Figure 3C). Microscopic observation showed that the WT and CP strains began to yield conidia at 18 h, while the ΔMaAreB strain did at 21 h ( Figure 3D). Furthermore, the conidial yield of the ∆MaAreB strain significantly reduced ( Figure 3E). Taken together, it revealed that the MaAreB contributed to the conidial germination and the conidiation of M. acridum.

Deletion of MaAreB Affected Conidial Germination, Hyphal Growth and Conidiation
To analyze the growth characteristics of the WT, ∆MaAreB and CP strains, we tested the conidial germinations, the macroscopic and microscopic growth morphologies, and the conidial yield of fungal strains grown on 1/4SDAY. The results showed that although there was no significant difference in colony morphology among the WT, ∆MaAreB and CP strains ( Figure 3A), the germination rate of ∆MaAreB strain was significantly slower at 2, 4, 6, 8 and 10 h ( Figure 3B). The GT 50 (the mean 50% germination time) of the ∆MaAreB strain (6.90 ± 0.23 h) was significantly prolonged compared with the WT (5.25 ± 0.20 h) or CP (5.43 ± 0.19 h) strain ( Figure 3C). Microscopic observation showed that the WT and CP strains began to yield conidia at 18 h, while the ∆MaAreB strain did at 21 h ( Figure 3D). Furthermore, the conidial yield of the ∆MaAreB strain significantly reduced ( Figure 3E). Taken together, it revealed that the MaAreB contributed to the conidial germination and the conidiation of M. acridum.  Figure 3D indicated the conidium formed at the tip of the hypha. A&B, A&C and B&C, p < 0.05 (Tukey's HSD). The p-values in Figure 3B, C and E are listed in supplementary Table S3.

Deletion of MaAreB Weakened the Stress Tolerances to UV-B Irradiation and Heat-Shock
To clarify the effect of MaAreB on stress tolerances, the conidial germination rates of fungal strains were determined after treating with UV-B and heat-shock. These results showed that the conidial germination rate of ∆MaAreB was sharply decreased under UV-B treatment at 2.50 h, 3.75 h and 5.00 h ( Figure 4A). The IT50 (mean 50% inhibition time) of the ∆MaAreB strain (3.65 ± 0.04 h) was markedly decreased compared with the WT (4.51 ± 0.16 h) or CP (4.34 ± 0.12 h) strain ( Figure 4B). Furthermore, the transcription of the UV-B tolerance-related genes, MaUve1, MaWC1 and MaPhr, were significantly decreased in ΔMaAreB ( Figure 4C). After being treated with heat-shock, the germination rate of the ∆MaAreB strain also decreased significantly ( Figure 4D). The IT50 of ∆MaAreB (9.62 ± 0.41 h) was significantly lower than that of the WT (12.26 ± 0.63 h) or CP (11.73 ± 0.53 h) strain ( Figure 4E). In addition, the MaUbi1 gene related to the heat-shock protection of fungus was significantly down-regulated in ∆MaAreB ( Figure 4F). In summary, MaAreB played critical roles in fungal tolerances to UV-B irradiation and heat-shock.  Figure 3D indicated the conidium formed at the tip of the hypha. A&B, A&C and B&C, p < 0.05 (Tukey's HSD). The p-values in Figure 3B,C,E are listed in supplementary Table S3.

Deletion of MaAreB Weakened the Stress Tolerances to UV-B Irradiation and Heat-Shock
To clarify the effect of MaAreB on stress tolerances, the conidial germination rates of fungal strains were determined after treating with UV-B and heat-shock. These results showed that the conidial germination rate of ∆MaAreB was sharply decreased under UV-B treatment at 2.50 h, 3.75 h and 5.00 h ( Figure 4A). The IT 50 (mean 50% inhibition time) of the ∆MaAreB strain (3.65 ± 0.04 h) was markedly decreased compared with the WT (4.51 ± 0.16 h) or CP (4.34 ± 0.12 h) strain ( Figure 4B). Furthermore, the transcription of the UV-B tolerance-related genes, MaUve1, MaWC1 and MaPhr, were significantly decreased in ∆MaAreB ( Figure 4C). After being treated with heat-shock, the germination rate of the ∆MaAreB strain also decreased significantly ( Figure 4D). The IT 50 of ∆MaAreB (9.62 ± 0.41 h) was significantly lower than that of the WT (12.26 ± 0.63 h) or CP (11.73 ± 0.53 h) strain ( Figure 4E). In addition, the MaUbi1 gene related to the heat-shock protection of fungus was significantly down-regulated in ∆MaAreB ( Figure 4F). In summary, MaAreB played critical roles in fungal tolerances to UV-B irradiation and heat-shock.

Disruption of MaAreB Affected the Cell Wall Integrity
The fungal cell wall plays crucial roles in fungal resistance to various adversities [42]. To further analyze the influence of MaAreB on the cell wall integrity of M. acridum, we added CFW into the 1/4SDAY to observe the growth of each strain. It was found that the colony of the ΔMaAreB strain was smaller than those of the WT and CP strains (Figure 5A), and the relative inhibition rate of the ΔMaAreB strain increased dramatically compared to the WT or CP strain ( Figure 5B). Furthermore, the hyphal growth of the ΔMaAreB strain was significantly inhibited by CFW ( Figure 5C). These results indicated that the disruption of MaAreB affected the cell wall integrity. Thus, the cell wall components of each strain were measured. As a result, the contents of chitin and β-1,3-glucan decreased significantly when the MaAreB gene was deleted, but the content of mannoproteins was not affected ( Figure 5D). Meanwhile, some genes related to cell wall integrity, such as chitin synthase genes MaChsⅠ, MaChsⅡ, MaChsⅢ, MaChsⅣ, MaChsⅤ , MaChs Ⅵ , MaChs Ⅶ and β-1,3-glucan synthase gene MaFks1, were significantly down-regulated in the ΔMaAreB strain ( Figure 5E). Apparently, these results revealed that disruption of MaAreB affected the cell wall composition and structure of M. acridum.

Disruption of MaAreB Affected the Cell Wall Integrity
The fungal cell wall plays crucial roles in fungal resistance to various adversities [42]. To further analyze the influence of MaAreB on the cell wall integrity of M. acridum, we added CFW into the 1/4SDAY to observe the growth of each strain. It was found that the colony of the ∆MaAreB strain was smaller than those of the WT and CP strains ( Figure 5A), and the relative inhibition rate of the ∆MaAreB strain increased dramatically compared to the WT or CP strain ( Figure 5B). Furthermore, the hyphal growth of the ∆MaAreB strain was significantly inhibited by CFW ( Figure 5CMaAreB affected the cell wall integrity. Thus, the cell wall components of each strain were measured. As a result, the contents of chitin and β-1,3-glucan decreased significantly when the MaAreB gene was deleted, but the content of mannoproteins was not affected ( Figure 5D). Meanwhile, some genes related to cell wall integrity, such as chitin synthase genes MaChsI, MaChsII, MaChsIII, MaChsIV, MaChsV, MaChsVI, MaChsVII and β-1,3-glucan synthase gene MaFks1, were significantly down-regulated in the ∆MaAreB strain ( Figure 5E). Apparently, these results revealed that disruption of MaAreB affected the cell wall composition and structure of M. acridum.

Disruption of MaAreB Decreased Virulence
Bioassays were performed to test the virulence of the fungal strains through topical inoculation and intra-hemocoel injection. The topical inoculation experiment showed that the survival rates of locusts infected by the ΔMaAreB strain were higher than those of the WT or CP strain at the same infection time, and visualization of locust cadavers killed by the ΔMaAreB strain indicated less conidiation at 8 dpi ( Figure 6A). The LT50 of the ΔMaAreB strain was 6.72 ± 0.11 d, which was significantly higher than that of the WT (6.08 ± 0.09 d) or CP (6.18 ± 0.08 d) strain ( Figure 6B). Furthermore, the quantification of the DNA concentrations of hyphal bodies in the locust hemolymph was performed by quantitative PCR. As a result, the fungal DNA concentrations in locusts infected by the ΔMaAreB strain were markedly decreased at 4 dpi and 6 dpi compared to those infected by the WT or CP strain via topical inoculation ( Figure 6C). Similarly, the number of hyphal bodies were also reduced greatly in those infected by the ΔMaAreB strain ( Figure  6D, E).
For the injection experiment, however, there was no significantly difference in the survival rates of locusts ( Figure S2A), the LT50s (Figure S2B), the fungal DNA concentra-

Disruption of MaAreB Decreased Virulence
Bioassays were performed to test the virulence of the fungal strains through topical inoculation and intra-hemocoel injection. The topical inoculation experiment showed that the survival rates of locusts infected by the ∆MaAreB strain were higher than those of the WT or CP strain at the same infection time, and visualization of locust cadavers killed by the ∆MaAreB strain indicated less conidiation at 8 dpi ( Figure 6A). The LT 50 of the ∆MaAreB strain was 6.72 ± 0.11 d, which was significantly higher than that of the WT (6.08 ± 0.09 d) or CP (6.18 ± 0.08 d) strain ( Figure 6B). Furthermore, the quantification of the DNA concentrations of hyphal bodies in the locust hemolymph was performed by quantitative PCR. As a result, the fungal DNA concentrations in locusts infected by the ∆MaAreB strain were markedly decreased at 4 dpi and 6 dpi compared to those infected by the WT or CP strain via topical inoculation ( Figure 6C). Similarly, the number of hyphal bodies were also reduced greatly in those infected by the ∆MaAreB strain ( Figure 6D,E). tions ( Figure S2C) and the number of hyphal bodies in locusts among all fungal strains ( Figure S2D, E). Taken together, these results indicated that loss of MaAreB severely affected the insect cuticle penetration process, reducing the virulence of M. acridum.  (Table S1) as described previously [39]. (D) The number of hyphal bodies in locust hemolymph by topical inoculation at 6 dpi. dpi, days post inoculation. (E) Microscopic images of hyphal bodies in locust hemolymph by topical inoculation at 6 dpi. The white arrows indicate the locust blood cells, and the black arrows indicate the hyphal bodies. A&B, p < 0.05 (Tukey's HSD). The p-values in Figure 6B-D are listed in supplementary Table S3. Furthermore, the conidial germinations and appressorium formations of fungal strains were determined on the locust hind wings. The conidial germination was accelerated with the deletion of MaAreB ( Figure 7A). The GT50 of the ΔMaAreB strain (5.07 ± 0.09 h) was significantly lower than that of the WT (6.62 ± 0.18 h) or CP (6.76 ± 0.15 h) strain ( Figure 7B). However, the appressorium formation of the ΔMaAreB strain (52.00%) was heavily decreased compared to that of the WT (72.67%) or CP (72.33%) strain after 24 h ( Figure 7C). qRT-PCR analyses showed that genes involved in fungal adhesion (MaMad1 and MaMad2) and cuticle penetration (MaPr1 and MaChit1) were significantly down-regulated in the ΔMaAreB strain ( Figure 7D). The data indicated that loss of MaAreB affected the conidial germination and appressorium formation on the locust hind wings and the expression of genes related to adhesion and cuticle penetration.  (Table S1) as described previously [39]. (D) The number of hyphal bodies in locust hemolymph by topical inoculation at 6 dpi. dpi, days post inoculation. (E) Microscopic images of hyphal bodies in locust hemolymph by topical inoculation at 6 dpi. The white arrows indicate the locust blood cells, and the black arrows indicate the hyphal bodies. A&B, p < 0.05 (Tukey's HSD). The p-values in Figure 6B-D are listed in supplementary Table S3. For the injection experiment, however, there was no significantly difference in the survival rates of locusts ( Figure S2A), the LT 50 s (Figure S2B), the fungal DNA concentrations ( Figure S2C) and the number of hyphal bodies in locusts among all fungal strains ( Figure S2D,E). Taken together, these results indicated that loss of MaAreB severely affected the insect cuticle penetration process, reducing the virulence of M. acridum.
Furthermore, the conidial germinations and appressorium formations of fungal strains were determined on the locust hind wings. The conidial germination was accelerated with the deletion of MaAreB ( Figure 7A). The GT 50 of the ∆MaAreB strain (5.07 ± 0.09 h) was significantly lower than that of the WT (6.62 ± 0.18 h) or CP (6.76 ± 0.15 h) strain ( Figure 7B). However, the appressorium formation of the ∆MaAreB strain (52.00%) was heavily decreased compared to that of the WT (72.67%) or CP (72.33%) strain after 24 h ( Figure 7C). qRT-PCR analyses showed that genes involved in fungal adhesion (MaMad1 and MaMad2) and cuticle penetration (MaPr1 and MaChit1) were significantly downregulated in the ∆MaAreB strain ( Figure 7D). The data indicated that loss of MaAreB affected the conidial germination and appressorium formation on the locust hind wings and the expression of genes related to adhesion and cuticle penetration. Turgor pressure is closely involved in the penetration of appressorium [9]. The appressorium collapsed rates of fungal strains were examined in different concentrations of PEG8000. The results showed that the appressorium collapsed rates of the ΔMaAreB strain were increased significantly compared with that from the WT or CP strain ( Figure  8A). The glycerol produced by lipids in appressorium can enhance turgor pressure and promote the penetration of appressorium [41,43,44]. qRT-PCR analyses showed that the transcription of glycerol synthesis-related genes, MaGPD1 and MaMPL1 decreased significantly in ΔMaAreB ( Figure 8B). Nile red staining demonstrated that the fluorescence intensity of the neutral lipids in appressoria of the ΔMaAreB strain was weaker than that in the WT or CP strain ( Figure 8C, D). Turgor pressure is closely involved in the penetration of appressorium [9]. The appressorium collapsed rates of fungal strains were examined in different concentrations of PEG8000. The results showed that the appressorium collapsed rates of the ∆MaAreB strain were increased significantly compared with that from the WT or CP strain ( Figure 8A). The glycerol produced by lipids in appressorium can enhance turgor pressure and promote the penetration of appressorium [41,43,44]. qRT-PCR analyses showed that the transcription of glycerol synthesis-related genes, MaGPD1 and MaMPL1 decreased significantly in ∆MaAreB ( Figure 8B). Nile red staining demonstrated that the fluorescence intensity of the neutral lipids in appressoria of the ∆MaAreB strain was weaker than that in the WT or CP strain ( Figure 8C,D).

Discussion
GATA transcription factors play vital roles in the NCR pathway, and regulate cell differentiation in different tissues [45]. The classical GATA transcription factor functions as a transcription activator and contains a transcription activation region at the N-terminal [45]. In filamentous fungi, only two GATA transcription factors, AreA and AreB, are found in the NCR pathway, among which AreA plays a positive regulatory role and AreB plays a repressive role [13,46]. AreA is a global transcriptional regulator related to nitrogen metabolism in filamentous fungi, which can activate the transcription of genes related to the secondary nitrogen metabolism, and it has been reported in many fungi, such as A. nidulans [47][48][49], A. parasiticus [50], Gibberella fujikuroi [51], F. verticillioides [26], and F. fujikuroi [18]. On the contrary, there are a few studies on AreB in filamentous fungi, which mainly focus on nitrogen metabolism and the regulatory relationship with NCR related genes [11,14,24]. In this study, we identified the AreB homologous protein, MaAreB, in the model insect pathogenic fungus M. acridum. The functions of MaAreB in nitrogen utilization, growth and development, tolerance to both, and virulence were characterized.
Based on the functions of GATA transcription factors in nitrogen metabolism, we explored the selective utilization of nitrogen sources by MaAreB. The results showed that the utilization ability of both preferential utilization glutamine and less easily assimilated nitrate were decreased in the ΔMaAreB strain. Although there was no significant difference in colony diameter on the ammonium, the conidial yield was significantly reduced in ΔMaAreB. These results suggested that MaAreB was also involved in regulating  Figure 8A,B,D are listed in supplementary Table S3.

Discussion
GATA transcription factors play vital roles in the NCR pathway, and regulate cell differentiation in different tissues [45]. The classical GATA transcription factor functions as a transcription activator and contains a transcription activation region at the N-terminal [45]. In filamentous fungi, only two GATA transcription factors, AreA and AreB, are found in the NCR pathway, among which AreA plays a positive regulatory role and AreB plays a repressive role [13,46]. AreA is a global transcriptional regulator related to nitrogen metabolism in filamentous fungi, which can activate the transcription of genes related to the secondary nitrogen metabolism, and it has been reported in many fungi, such as A. nidulans [47][48][49], A. parasiticus [50], Gibberella fujikuroi [51], F. verticillioides [26], and F. fujikuroi [18]. On the contrary, there are a few studies on AreB in filamentous fungi, which mainly focus on nitrogen metabolism and the regulatory relationship with NCR related genes [11,14,24]. In this study, we identified the AreB homologous protein, MaAreB, in the model insect pathogenic fungus M. acridum. The functions of MaAreB in nitrogen utilization, growth and development, tolerance to both, and virulence were characterized.
Based on the functions of GATA transcription factors in nitrogen metabolism, we explored the selective utilization of nitrogen sources by MaAreB. The results showed that the utilization ability of both preferential utilization glutamine and less easily assimilated nitrate were decreased in the ∆MaAreB strain. Although there was no significant difference in colony diameter on the ammonium, the conidial yield was significantly reduced in ∆MaAreB. These results suggested that MaAreB was also involved in regulating nitrogen utilization in M. acridum. Furthermore, loss of MaAreB resulted in a delay in conidial germination and a decrease in conidial yield when fungal strains were grown on the complete medium (1/4SDAY), indicating that MaAreB was involved in regulating the development and asexual sporulation.
Metarhizium spp. is a kind of important biocontrol fungus, which can infect a variety of agricultural pests [52][53][54][55][56]. The adaptability of entomopathogenic fungi to the adverse environment directly determine the efficacy of mycoinsecticides [57]. In this study, we found that loss of MaAreB reduced the tolerances to UV-B irradiation and heat-shock in M. acridum. As a powerful physiological barrier, the fungal cell wall involves a variety of physiological functions, such as nutrient uptake and cell attachment, maintaining the inherent morphology of fungal cells, and protecting cells from external adversity conditions [58][59][60][61]. Our results showed that MaAreB affected the cell wall integrity of M. acridum. The hyphal growth and colony morphology of the ∆MaAreB strain were seriously inhibited by CFW, which could bind to the nascent chitin chain to prevent the formation of co-crystals and microfibrils [62]. Chitin is synthesized by chitin synthases and is crucial to the fungal cell wall integrity [63]. Studies have shown that ChsIII, ChsV, ChsVI and ChsVII affect the synthesis of polysaccharide in the cell wall, and ChsIII, ChsV and ChsVII are necessary for the cell wall integrity in M. acridum [36]. In addition, chitin syn synthases in A. fumigatus [64] and Botrytis cinerea [65] also affect the cell wall integrity. We found that the contents of chitin and β-1,3-glucan, the essential components of the fungal cell wall, in ∆MaAreB strain were heavily decreased. Consistently, some genes encoding chitin synthases or β-1,3-glucan synthase were significantly down-regulated. Therefore, we speculated that MaAreB might make contributions to the fungal sensitivity and resistance to the external environment by affecting the cell wall integrity.
Studies in many fungi, such as Ustilago maydis [66], M. oryzae [67], and A. fumigatus [64,68], have pointed out that the change of chitin content in the cell wall affects appressorium formation, which in turn affects the host immune defense. In this study, we found that disruption of MaAreB affected the synthesis of fungal cell wall components and cell wall integrity. The virulence test showed that the virulence of the ∆MaAreB strain was significantly decreased in topical inoculation, while it was not significantly different from that of the WT or CP strain in intra-hemocoel injection, indicating that MaAreB was conducive for M. acridum to penetrate the host cuticle. The ability of conidium to penetrate the host cuticle was directly determined by its adhesion, germination, and appressorium formation on the host cuticle [8]. Our study proved that the appressorium formation, the appressorium turgor pressure, and the transcription of genes related to adhesion (MaMad1 and MaMad2) and penetration (MaPr1 and MaChit1) were all significantly reduced in the ∆MaAreB strain. Appressoria of entomopathogenic fungi are necessary for infecting host, and the turgor pressure and cuticle degrading enzymes can promote appressoria to penetrate the host cuticle [9]. These data further confirmed that MaAreB played an important role in insect cuticle penetration of M. acridum.
Nitrogen metabolism is not only involved in regulating the nutrient utilization of fungi, but also closely involved in the virulence of pathogenic fungi. Glutamine, a preferential nitrogen source, could inhibit the virulence of M. oryzae [28,69] and F. oxysporum [70]. Deletion of MoGln1, the glutamine synthetase gene, would affect the virulence of infecting hyphae in M. oryzae [69]. During the process of host infection by entomopathogenic fungi, conidial germination [71,72] and appressorium formation [73] are closely related to nitrogen source conditions. B. bassiana needs to activate its own intracellular nitrogen metabolism level to promote conidial germination; if not, the conidia cannot form germ tubes [74]. Nitrogen starvation can promote appressorium formation in M. anisopliae [75]. Nitrogen starvation can induce the expression of many pathogenesis related genes; hence it is also regarded as the driving force for pathogenic fungi to infect the host [76,77]. In M. oryzae, deletion of Asd4, the AreB homologous gene, could increase glutamine content and activate TOR (Target of Rapamycin) pathway, and result in the inability to form appressorium [28]. However, the appressorium formation could be restored with knocking out Gln1 or inhibiting TOR path under ∆Asd4 background [28]. In M. acridum, MaAreB was involved in regulating nitrogen metabolism and virulence, but it was still unclear whether there was a certain correlation between nutrient utilization and virulence. Whether MaAreB affects virulence by inducing nitrogen metabolism remains to be explored in the near future.

Conclusions
Nutrients are very important for fungal growth and development. It is well known that the NCR pathway plays crucial roles in the regulation of fungal nitrogen metabolism. During these processes, GATA-type transcription factors, such as AreA and AreB, play indispensable roles. Although AreA has been reported in many fungi, the second GATA transcription factor AreB is less studied than AreA. The functions of AreB are largely unknown in entomopathogenic fungi. In this study, we focused on functional characterization of the second GATA transcription factor MaAreB in the model entomopathogenic fungus M. acridum. Deletion of MaAreB affected the fungal cell wall integrity and reduced fungal virulence due to weakening the cuticle penetration of M. acridum. However, the roles of MaAreB in nitrogen utilization needs to be further investigated in M. acridum. In general, these results provide insights into understanding the functions of AreB homologous proteins in other filamentous fungi.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jof7070512/s1, Figure S1: Vector construction and Southern blotting. Schematic diagram of MaAreB knockout (A) and complemented (B) vector constructions. (C) Verification of the WT, ∆MaAreB and CP strains by Southern blotting. Probe was amplified with primers AreB-PF/AreB-PR (Table S1). Restriction enzyme EcoRV and SacII were used to digest the genome DNA of the WT, ∆MaAreB and CP strains. Figure S2: Loss of MaAreB did not affected the virulence in intra-hemocoel injection. (A) Survival rates of locusts infected with the WT, ∆MaAreB and CP strains in intrahemocoel injection, respectively. Locust cadavers killed by fungal strains were cultured at 28 • C for 8 days. (B) LT 50 s of fungal strains in intra-hemocoel injection test. (C) DNA concentrations of M. acridum in hemolymph of locust infected with the WT, ∆MaAreB and CP strains for 1 d, 3 d and 5 d in intra-hemocoel injection test. For each treatment, 600 µL blood was taken from 20 locusts (30 µL for a locust) to extract the genomic DNA. The fungal DNA concentrations were determined using quantitative PCR with primer pair of ITS-F/ITS-R (Table S1) as described previously [39]. (D) The number of hyphal bodies in locust hemolymph by intra-hemocoel injection at 5 dpi. (E) Microscopic images of hyphal bodies in locust hemolymph by intra-hemocoel injection at 5 dpi. dpi, days post inoculation. The white arrows indicate the locust blood cells, and the black arrows indicate the hyphal bodies. A&A, p > 0.05 (Tukey's HSD). The p values in Figure S2B-D were listed in supplementary Table S3. Table S1: Primers used in this study. Table S2: Parameter information of primer pairs used in qRT-PCR.

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