ChsA, a Class Ⅱ Chitin Synthase, Contributes to Asexual Conidiation, Mycelial Morphology, Cell Wall Integrity, and the Production of Enzymes and Organic Acids in Aspergillus niger

Chitin synthases (CHSs) are vital enzymes for the synthesis of chitin and play important and differential roles in fungal development, cell wall integrity, environmental adaptation, virulence, and metabolism in fungi. However, except for ChsC, a class III CHS, little is known about the functions of CHSs in Aspergillus niger, an important fungus that is widely applied in the fermentation industry and food processing, as well as a spoilage fungus of food and a human pathogen. This study showed the important functions of ChsA, a class II CHS, in A. niger using multi-phenotypic and transcriptional analyses under various conditions. The deletion of chsA led to severe defects in conidiation on different media and resulted in the formation of smaller and less compact pellets with less septa in hyphal cells during submerged fermentation. Compared with the WT, the ΔchsA mutants exhibited less chitin content, reduced growth under the stresses of cell wall-disturbing and oxidative agents, more released protoplasts, a thicker conidial wall, decreased production of amylases, pectinases, cellulases, and malic acid, and increased citric acid production. However, ΔchsA mutants displayed insignificant changes in their sensitivity to osmotic agents and infection ability on apple. These findings concurred with the alteration in the transcript levels and enzymatic activities of some phenotype-related genes. Conclusively, ChsA is important for cell wall integrity and mycelial morphology, and acts as a positive regulator of conidiation, cellular responses to oxidative stresses, and the production of malic acid and some enzymes, but negatively regulates the citric acid production in A. niger.


Introduction
Chitin, the β-1,4-linked polymer of N-acetylglucosamine, is a key cell wall component of fungi and is essential for cell wall organization and integrity, which play important roles in the protection for cells [1,2].The core of the chitin biogenesis machinery is chitin synthases (CHSs), which catalyze the polymerization of the monomer N-acetyl-D-glucosamine to produce single nascent chitin chains that assemble into chitin nanofibers once they have been secreted [2].In Saccharomyces cerevisiae, three CHS-encoding genes designated as Chs1, Chs2, and Chs3 have been identified which play roles in the repairment of damaged chitin during cell separation, primary septum formation, and all other chitin syntheses, respectively [3].Moreover, cells lacking chs1 grew normally and showed insignificant changes in the cell wall chitin content, but the disruption of chs2 resulted in severe growth defects and morphological abnormalities [3].In filamentous fungi, there are usually approximately metabolites, such as organic acids, bioactive compounds, and enzymes; to improve the sensory characteristics of fermented foods and bioavailability of polyphenols; and to detoxify some mycotoxins such as aflatoxin B1, which have great economic and research significance [12][13][14][15].Additionally, A. niger could cause rot in vegetables and fruits, which leads to a huge waste of resources [16].More seriously, A. niger might cause pulmonary aspergillosis, which is harmful to human health [17].Therefore, in order to improve the fermentation performance of, and reduce contamination or infection by A. niger, it is necessary to unveil its molecular mechanisms which are involved in the regulation of fungal growth and development, metabolism, and pathogenicity.There exist eight CHSencoding genes in the genome database of A. niger [15], and the silencing of chsC, a class III CHS-encoding gene, resulted in a reduction in conidiation efficiency and the chitin content of the cell wall, as well as increases in the compactness of the mycelial pellets and the production of citric acid [14], confirming the indispensability of the chitin synthase in conidiation, mycelial morphology, and the production of citric acid in A. niger.These results suggest that other CHS-encoding genes might play vital roles in fungal development and metabolism of A. niger.Moreover, previous studies have shown that chsA (a class II CHS-encoding gene) and chsC might be the explanation for the formation of dispersed mycelia rather than the pellets caused by epigallocatechin-3-gallate (EGCG) in A. niger RAF106, which might also result in changes in the production of some metabolites such as cellulases and pectinases [18].However, little is known about the roles of chsA in A. niger.Therefore, this study aimed to elucidate the functions of A. niger chsA through analysis of the changes in multi-phenotypes, the production of various enzymes and organic acids, and the transcriptional expression of phenotype-related genes between the wild-type strain and chsA deletion mutants.The results demonstrated that the chsA gene was closely associated with asexual conidiation, mycelial morphology, cell wall integrity, cellular response to oxidative stresses, and the production of some enzymes and organic acids, which will be helpful in enriching the understanding of the Class II CHSs, improving their fermentation performance, and controling the contamination of A. niger.

Cloning and Analysis of chsA in A. niger
The sequence of chsA (gene08422) was amplified from the WT using paired primers (Table S1) and was sequenced at Invitrogen.The deduced protein sequence was structurally analyzed via an online BLAST available online: http://blast.ncbi.nlm.nih.gov/blast.cgi(accessed on 17 June 2023), and phylogenetically analyzed with class II chitin synthases from other fungi using a neighbor-joining method in MEGA 7.0 software (Mega Limited, Auckland, New Zealand).The molecular size and theoretical isoelectric point of ChsA were predicted using the available online: https://web.expasy.org/protparam/(accessed on 20 June 2023) and the subcellular localization was predicted using the available online: https://wolfpsort.hgc.jp/(accessed on 20 June 2023).

Construction of chsA Mutants
ChsA was deleted by homologous replacement [20].Briefly, the expression cassette, including the promoter and open reading frame of hygB (conferring resistance to hygromycin B), was amplified from pSilent-1 with paired primers (Table S1) and cloned into the p0380bar cut with SacI/XhoI, forming p0380-hygB.The chsA disruption vector was constructed by cloning the 5 and 3 fragments of the gene amplified from the WT genome via PCR with paired primers (Table S1) into p0380-hygB cut with XmaI/SmaI and XhoI/XbaI, respectively.The disruption plasmid was introduced into WT via Agrobacterium-mediated transformation [19].Putative mutant colonies obtained from a selective medium with cefotaxime (300 µg/mL) and hygromycin B (50 µg/mL) were identified via PCR and quantitative real-time PCR (qRT-PCR) with paired primers (Table S1).

Determination of Fungal Growth and Conidiation
A suspension of 1 µL of 1 × 10 6 conidia/mL was spotted on plates (9 cm diameter) of SDAY, CZA, and PDA media.After a 2-day incubation at 30 • C, the diameter of each colony was cross-measured as an index of fungal growth.To assess the capacity for conidiation, a suspension of 100 µL of 1 × 10 6 conidia/mL was spread evenly on SDAY, CZA, and PDA plates and incubated at 30 • C. From day 3, colony discs (5 mm diameter) were cut randomly from the plates with a cork borer and individually immersed in 1 mL of 0.02% Tween 80 solution by supersonic vibration for 10 min.The suspension was filtered through four layers of lens tissues to remove hyphal debris, and the conidial concentration was measured using a hemocytometer and then converted to the number of conidia per cm 2 of the colony.Meanwhile, 3-day cultures were cut using a razor blade and observed under a microscope to assess the morphological changes in conidiophores.

Assays for Pellet Formation and Condidial Agglomeration
A suspension of 1 mL of 1 × 10 8 conidia/mL was added into 100 mL of potato dextrose broth (PDB; agar-free PDA) and shaken at 180 rpm for 48 h at 30 • C.During the first 4 h incubation, conidial agglomeration was observed under a microscope at 2 h intervals.Morphological changes in fungal mycelia were examined under a microscope at 12 h intervals.

Assessments for Cellular Responses to Chemical Stresses
A suspension of 1 µL of 10 6 spores/mL was spotted on plates (9 cm diameter) of PDA alone (control) and PDA media supplemented with different concentrations of Congo red (CR; 2 mg/mL and 3 mg/mL), sodium dodecyl sulfate (SDS; 0.03%, and 0.04%), NaCl (1.2 M), KCl (1.2 M), menadione (MND; 1 µM and 2 µM), and H 2 O 2 (6 mM and 12 mM), respectively.After a 2-day incubation at 30 • C, the diameter of each colony was measured as mentioned and the relative growth inhibition was calculated as (D c − D t )/D c × 100%, where D c and D t are colony diameters on the control plates and tested plates with the responding chemical stress for each strain, respectively.

Examination of Cell Wall Changes
Cell wall isolation and chitin content analysis were examined as described previously [21].Briefly, a suspension of 1 mL of 10 8 spores/mL was added into 100 mL of complete liquid medium (CM; 1% glucose, 0.1% tyrosine, and 0.5% yeast extract) and shaken at 180 rpm at 30 • C.After a 17 h incubation, mycelia were harvested, ground in liquid nitrogen, and washed with 50 mL of 1 M NaCl and 50 mL of high-quality deionized water (MQ) to remove intracellular debris and proteins.The cell wall suspension was centrifuged at 5000 rpm for 10 min to obtain the cell wall.Chitin in the cell wall was measured as total glucosamine based on the principle of the Morgan-Elson protocol [22].
The fragility of the cell wall was assayed as described previously with slight modification [23].Briefly, a suspension of 1 mL of 10 8 spores/mL was added into 100 mL of Sabouraud dextrose broth (SDB; agar-free SDAY) and shaken at 180 rpm at 30 • C.After a 17 h incubation, mycelia (100 mg) were harvested, washed twice with 0.1 M phosphate buffer (PBS; pH 6.8), resuspended in 2 mL of 0.8 M KCl containing 0.1% snailase (Beyotime, Shanghai, China) and 1.5% lysing enzyme (Beyotime, Shanghai), and incubated at 37 • C for 3.5 h with shaking at 150 rpm.The concentration of protoplasts released from the hyphal cells was measured using a hemocytometer plate and considered as an index of cell wall fragility.
The cell wall of aerial conidia was observed under transmission electron microscopy (TEM) as described previously [24].Briefly, conidia were harvested from 5-day cultures cultivated on the PDA plates, washed with PBS, fixed in the 2.5% glutaraldehyde buffer, washed with PBS, dehydrated in a gradient ethanol series, embedded in Spurr resin, dyed with uranyl acetate solution and a lead solution supplemented with citric acid, and observed under a Talos L120C TEM (FEI).

Calcofluor White Staining
Calcofluor white (CFW) staining of the hyphae was performed as described previously [25].Briefly, a suspension of 1 mL of 10 8 spores/mL was added into 100 mL PDB with 50 glass beads and shaken at 180 rpm at 30 • C.After a 24 h incubation, the hyphae were collected and placed on the cover slide containing a 20 µL droplet of 10 µg/mL CFW.After a 5 min incubation, the CFW fluorescence was observed under a fluorescence microscope (Axio Observer A1, Carl Zeiss, Oberkochen, Germany).

Quantitative Analysis of Antioxidant Enzymes
Aliquots of 100 mg hyphal mass from 3-day-old PDA cultures grown at 30 • C were ground in liquid nitrogen, re-suspended in PBS buffer, and centrifuged at 4 • C. The supernatant was collected and served as the crude enzymes.The activities of superoxide dismutases (SODs) and catalases (CATs) were measured by total superoxide dismutase assay kit with WST-8 (S0101S) and catalase assay kit (S0051), respectively, and the protein concentration was measured by BCA Protein Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's directions.The number of SODs and CATs was measured at 450 and 520 nm, respectively, under a microplate reader (Molecular Devices VersaMax, San Jose, CA, USA).One unit of SOD activity was defined as the SOD amount required to inhibit 50% of the coupling reaction of xanthine.One unit of CAT activity was defined as 1 µmol H 2 O 2 consumed per minute at 25 • C and pH 7.0.

Assays for Amylases, Cellulases, Pectinases and Proteases
The production of amylases, cellulases, and pectinases was measured as described previously [18].Briefly, a suspension of 1 mL of 10 8 spores/mL was added into 100 mL Wheat Bran straw medium (WBS; 55 g/L rice straw flour, 1.6 g/L (NH 4 ) 2 SO 4 , 0.2 g/L MgSO 4 , 30 g/L peptone, 2 g/L KH 2 PO 4 , 0.01 g/L CaCl 2 , 0.002 g/L ZnCl 2 , 0.2 g/L urea, pH 5.0).After 3-day incubation with shaking at 30 • C, the supernatant was collected and served as the crude enzymes.For cellulase activity, 0.05 mL of crude enzymes were incubated with 0.95 mL of 1% sodium salt carboxy methyl-cellulose (CMC-Na) solution in 0.05 M sodium citrate buffer at 50 • C for 20 min.For amylase activity, 0.05 mL of crude enzymes were incubated with 0.45 mL of 1% starch solution in 0.05 M sodium citrate buffer at 50 • C for 30 min.For pectinase activity, 0.05 mL of crude enzymes were incubated with 0.45 mL of 0.5% pectin solution in 0.05 M sodium citrate buffer at 50 • C for 30 min.The reactions were stopped by adding 0.75 mL of 3,5-dinitro salicylic acid (DNS) reagent and immersing the reaction tube in boiling water (100 • C) for 10 min.One unit of enzymatic activity was defined as the amount of enzyme required to produce 1 mmol of D-glucose for cellulase activity, 1 mg of D-glucose for amylase activity, and 1 mg of D-galacturonic acid for pectinase activity by hydrolyzing the raw substrate per minute under one of the specified assay conditions.
The protease production was measured as described previously [26].Briefly, the supernatant was collected from 3-day cultures of A. niger incubated in WBS and served as the crude enzymes.Then, 0.5 mL of crude enzymes were incubated at 40 • C for 5 min and mixed with 1 mL of 2% casein which was incubated at 40 • C for a while.After a 10 min incubation at 40 • C, the reaction was stopped by adding 2 mL of 0.4 M trichloroacetic acid solution.After a 15 min incubation, the supernatant was collected, supplemented with 5 mL of 0.4 M sodium carbonate and 1 mL of 2 M Folin-phenol solution, and incubated at 40 • C for 20 min.The absorbance at 680 nm was measured and one unit of enzymatic activity was defined as the amount of enzyme required to produce 1 µg of tyrosine by hydrolyzing the raw substrate per minute under one of the specified assay conditions.

HPLC Analysis of Citric and Malic Acid Production
The production of citric and malic acid was analyzed using HPLC as described previously [27].Briefly, a suspension of 1 mL of 10 8 spores/mL was added into 100 mL WBS.After 3-day incubation with shaking at 30 • C, the supernatant was collected and filtered through membrane filters (0.22 µm).Aliquots of 10 µL of filtered supernatant were injected into an HPLC system equipped with a YMC C18 column (150 mm × 4.6 mm, 5 µm) and a UV detector set at 280 nm and eluted with the mobile solvent consisting of 10% methanol at a constant flow rate of 0.5 mL/min at 30 • C. The concentration of citric and malic acid was determined using external calibration curves of the standard solution at different concentrations.

qRT-PCR
Cultures for transcriptional expression of conidiation-related genes were initiated by spreading aliquots of 1 mL of 10 6 spores/mL on cellophane-attached PDA plates.After a 1-day incubation at 30 • C, mycelia were harvested, ground in liquid nitrogen, and used to extract total RNA under the action of a TRIzol ® Reagent (Invitrogen, Carlsbad, CA, USA).Total RNA was transcribed reversely into cDNA with a PrimeScript RT reagent kit (TaKaRa, Kyoto, Japan).Tenfold dilutions of each cDNA were used as templates to analyze the expression of tested genes via qRT-PCR with paired primers (Table S2) using SYBR ® Premix Ex Taq™ (TaKaRa, Kyoto, Japan).β-tubulin was used as an internal standard.The relative transcription level of each gene was defined as the ratio of its transcript in each mutant over that in WT using the 2 −∆∆Ct method [28].

Assay for the Infection Ability of A. niger on Apple
Healthy apples (175 g/apple) with uniform size, color, and weight were washed with tip water, and the surface was sterilized with 75% ethanol.Four wounds (2 mm in diameter and 3 mm deep) were made on the surface of each apple with a sterile yellow tip.After air-drying, aliquots of 5 µL of conidial suspension (final concentration of 10 6 spores/mL) were injected into each wound.The apples were placed in sealed containers and incubated continuously at 25 • C. The wound lesion was observed and photographed every day.

Statistical Analysis
All the experiments were conducted in triplicate.Results from replicates were expressed as mean ± standard deviation (SD) and the data analysis was subjected to a one-factor analysis of variance, followed by Tukey's HSD test.p < 0.05 was considered a significant difference in all experiments.

Bioinformatic Characteristics of ChsA in A. niger
A single ChsA sequence (Gene ID: gene08422) was found in the genome database of A. niger RAF106.Three introns consisting of 56, 60, and 51 bases, respectively, were found by comparing the genomic DNA and cDNA sequences.The open reading frame of chsA consists of 3036 bases, which encode 1011 amino acids with a molecular weight of approximately 112.48 kDa and an isoelectric point of 8.01.ChsA was predicted to locate in the plasma membrane and featured typical domains of CHS family members named Chitin_synth_1 (pfam01644) and Chitin_synth_C (cd04190), as well as three other conserved domains including the BcsA superfamily, which is the catalytic subunit of cellulose synthase and poly-beta-1,6-N acetylglucosamine synthase, Caps_synth_CapC superfamily, which is essential for the production of gamma-polyglutamic acid, and PHA03291 superfamily, which envelopes glucoprotein I (Figure 1A).Comparisons of the amimo acid sequences with other 24 CHSs belonging to the seven classes (from class I to VII) from A. fumigatus, A. nidulans, and Aspergillus luchuensis, as well as phylogenetic analysis, revealed that ChsA was subdivided into the Class II clade (Figure S1A).Moreover, phylogenetic analysis demonstrated that ChsA shares 42.07-99.59% of its sequence identity with 15 other class II CHSs found in fungi; it shows the highest similarity (99.59%) to a class 2 chitin synthase found in A. luchuensis, and the lowest similarity (42.07%) to Chs1, found in in Pyricularia oryzae (Figure 1B).
by comparing the genomic DNA and cDNA sequences.The open reading frame of chsA consists of 3036 bases, which encode 1011 amino acids with a molecular weight of approximately 112.48 kDa and an isoelectric point of 8.01.ChsA was predicted to locate in the plasma membrane and featured typical domains of CHS family members named Chi-tin_synth_1 (pfam01644) and Chitin_synth_C (cd04190), as well as three other conserved domains including the BcsA superfamily, which is the catalytic subunit of cellulose synthase and poly-beta-1,6-N acetylglucosamine synthase, Caps_synth_CapC superfamily, which is essential for the production of gamma-polyglutamic acid, and PHA03291 superfamily, which envelopes glucoprotein I (Figure 1A).Comparisons of the amimo acid sequences with other 24 CHSs belonging to the seven classes (from class I to VII) from A. fumigatus, A. nidulans, and Aspergillus luchuensis, as well as phylogenetic analysis, revealed that ChsA was subdivided into the Class II clade (Figure S1A).Moreover, phylogenetic analysis demonstrated that ChsA shares 42.07-99.59% of its sequence identity with 15 other class II CHSs found in fungi; it shows the highest similarity (99.59%) to a class 2 chitin synthase found in A. luchuensis, and the lowest similarity (42.07%) to Chs1, found in in Pyricularia oryzae (Figure 1B).

Effects of the Deletion of chsA on Radial Growth, Conidiation, and Mycelial Morphology
The deletion of chsA was conducted in the WT by homogeneous replacement via Agrobacterium-mediated transformation (Figure S1B).Three expected disruption mutants were confirmed by PCR and qRT-PCR analyses (Figure S1C), which demonstrated that chsA is nonessential for fungal viability in A. niger.
After a two-day incubation on PDA, SDAY, and CZA plates, all of the disruption mutants of chsA exhibited similar diameters of fungal colonies to the WT (Figure 2A).

Effects of the Deletion of chsA on Radial Growth, Conidiation, and Mycelial Morphology
The deletion of chsA was conducted in the WT by homogeneous replacement via Agrobacterium-mediated transformation (Figure S1B).Three expected disruption mutants were confirmed by PCR and qRT-PCR analyses (Figure S1C), which demonstrated that chsA is nonessential for fungal viability in A. niger.
However, the deletion of chsA caused severe defects in the conidiation capacity.Compared with the WT estimates of three-, four-, and five-day-old cultures, the mean yields of conidia in ΔchsA were reduced by 22.87-37.75%,12.55-18.37%,and 29.23-38.85%,respectively, for the PDA cultures (Figure 2B), 28.53-34.43%,68.38-72.10%,and 56.13-68.79%,respectively, for the SDAY cultures (Figure 2C), and 30.41-53.56%,38.57-49.58%,and 52.21-59.00%,respectively, for the CZA cultures (Figure 2D).Accompanied by the defects in conidial yields, ΔchsA showed sparser and smaller conidiophores than the WT according to the images observed under a microscope of samples obtained from the colonies on day three (Figure S2).Moreover, the transcripts of conidiation-required genes, including fluG, sfgA, flbA, flbC, flbE, laeA, brlA, vosA, and velB, were suppressed by 28.26-85.42% in ΔchsA compared with those in the WT when grown on PDA plates (Figure 2E).Additionally, the images analysis demonstrated that the deletion of chsA resulted in the formation of smaller and less compact pellets during submerged fermentation in PDB Additionally, the images analysis demonstrated that the deletion of chsA resulted in the formation of smaller and less compact pellets during submerged fermentation in PDB media, compared with the WT (Figure 3A).Correspondingly, the deletion of chsA inhibited conidial agglomeration.For the WT, a larger number of conidia aggregated after a 4 h incubation, and more conidia aggregated, germinated, and clustered in the group after 8 h incubation; however, for the deletion mutants of chsA, only a few conidia aggregated after 4 h incubation and the clusters of conidia and hyphae were smaller than that in the WT after 8 h incubation (Figure 3A).Moreover, CFW staining demonstrated that the number of the septa in the hyphae of ∆chsA was less than that in the WT (Figure 3B).These findings demonstrated that chsA participated in the maintenance of mycelial morphology during submerged fermentation.media, compared with the WT (Figure 3A).Correspondingly, the deletion of chsA inhibited conidial agglomeration.For the WT, a larger number of conidia aggregated after a 4 h incubation, and more conidia aggregated, germinated, and clustered in the group after 8 h incubation; however, for the deletion mutants of chsA, only a few conidia aggregated after 4 h incubation and the clusters of conidia and hyphae were smaller than that in the WT after 8 h incubation (Figure 3A).Moreover, CFW staining demonstrated that the number of the septa in the hyphae of ΔchsA was less than that in the WT (Figure 3B).These findings demonstrated that chsA participated in the maintenance of mycelial morphology during submerged fermentation.

Essential Roles of chsA in Cell Wall Integrity
The deletion of chsA led to remarkable cell wall damage.Compared with the WT, the deletion mutants of chsA exhibited a 77.61-84.26%decrease in the mean content of Nacetylglucosamine (Figure 4A), suggesting that chsA positively contributed to the chitin content in A. niger.Moreover, ΔchsA grew much slower than the WT under the stresses of 2 and 3 mg/mL Congo red and also showed a slight decrease in the diameter of the colony under the stress of 0.04% SDS, compared with that in the WT (Figure 4B).Interestingly, more protoplasts were released from hyphal cells after a 3.5 h incubation with cell lysing enzymes in ΔchsA than that in the WT (Figure 4C).Compared with the WT, the number of released protoplasts increased by 1.92-2.69-foldon average in ΔchsA, suggesting that the deletion of chsA led to an increase in the fragility of the hyphal wall.Furthermore, the TEM images showed that the outermost layer of the conidial wall became looser and thicker in all of the disruption mutants than that in the WT (Figure 4D).These results strongly indicated that chsA played important roles in the development of the cell wall in A. niger.

Essential Roles of chsA in Cell Wall Integrity
The deletion of chsA led to remarkable cell wall damage.Compared with the WT, the deletion mutants of chsA exhibited a 77.61-84.26%decrease in the mean content of N-acetylglucosamine (Figure 4A), suggesting that chsA positively contributed to the chitin content in A. niger.Moreover, ∆chsA grew much slower than the WT under the stresses of 2 and 3 mg/mL Congo red and also showed a slight decrease in the diameter of the colony under the stress of 0.04% SDS, compared with that in the WT (Figure 4B).Interestingly, more protoplasts were released from hyphal cells after a 3.5 h incubation with cell lysing enzymes in ∆chsA than that in the WT (Figure 4C).Compared with the WT, the number of released protoplasts increased by 1.92-2.69-foldon average in ∆chsA, suggesting that the deletion of chsA led to an increase in the fragility of the hyphal wall.Furthermore, the TEM images showed that the outermost layer of the conidial wall became looser and thicker in all of the disruption mutants than that in the WT (Figure 4D).These results strongly indicated that chsA played important roles in the development of the cell wall in A. niger.

Contribution of chsA to Chemincal Stresses and Virulence in A. niger
The deletion of chsA caused increases in sensitivities to the oxidants menadione and H2O2 during colony growth (Figure 5A).Compared with the estimates in the WT, the percentage of relative growth inhibition increased by 12.85-63.79%under the stresses of 1 and 2 µM menadione, respectively, and 14.34-19.78%under the stresses of 6 and 12 mM H2O2, respectively.Correspondingly, the SODs and CATs activities were lowered by 51.00-73.82%and 39.07-39.44% in ΔchsA, compared with those in the WT (Figure 5B,C).However, ΔchsA displayed similar growth to the WT under the stresses of 1.2 M NaCl and KCl and a similar lesion diameter to the WT on the apples (Figure 5A,D).These findings indicate that chsA was a positive regulator for cellular tolerance to the oxidants but was not involved in the fungal response to osmotic stresses and fungal pathogenicity in A. niger.

Contribution of chsA to Chemincal Stresses and Virulence in A. niger
The deletion of chsA caused increases in sensitivities to the oxidants menadione and H 2 O 2 during colony growth (Figure 5A).Compared with the estimates in the WT, the percentage of relative growth inhibition increased by 12.85-63.79%under the stresses of 1 and 2 µM menadione, respectively, and 14.34-19.78%under the stresses of 6 and 12 mM H 2 O 2 , respectively.Correspondingly, the SODs and CATs activities were lowered by 51.00-73.82%and 39.07-39.44% in ∆chsA, compared with those in the WT (Figure 5B,C).However, ∆chsA displayed similar growth to the WT under the stresses of 1.2 M NaCl and KCl and a similar lesion diameter to the WT on the apples (Figure 5A,D).These findings indicate that chsA was a positive regulator for cellular tolerance to the oxidants but was not involved in the fungal response to osmotic stresses and fungal pathogenicity in A. niger.

Changes in the Production of Amylases, Pectinases, Proteases, Cellulases, and Organic Acids
The deletion of chsA affected the production of amylases, pectinases, cellulases, citric acid, and malic acid rather than the production of proteases.Compared with those in the WT, the activities of amylases, pectinases, and cellulases were lowered by 56.48-61.95%,26.72-29.06%,and 52.85-60.19%,respectively, in the disruption mutants of chsA, but ΔchsA exhibited similar protease activity to the WT (Figure 6A-D).Moreover, ΔchsA displayed a 22.11-24.88%decrease in the production of malic acid but a 56.09-62.00%increase in the production of citric acid (Figure 6E,F).The deletion of chsA affected the production of amylases, pectinases, cellulases, citric acid, and malic acid rather than the production of proteases.Compared with those in the WT, the activities of amylases, pectinases, and cellulases were lowered by 56.48-61.95%,26.72-29.06%,and 52.85-60.19%,respectively, in the disruption mutants of chsA, but ∆chsA exhibited similar protease activity to the WT (Figure 6A-D).Moreover, ∆chsA displayed a 22.11-24.88%decrease in the production of malic acid but a 56.09-62.00%increase in the production of citric acid (Figure 6E,F).

Discussion
In fungi, chitin synthases play important but differential roles in cell wall integrity, cellular growth and development, environmental adaptation, metabolism, and pathogenicity [1,6,7,10,11,14,[29][30][31].In this study, A. niger ChsA, a class II chitin synthase, was proven to be involved in conidiation, mycelial morphology, cell wall integrity, cellular response to oxidative stress, and the production of enzymes and organic acids, as discussed below.
Firstly, ChsA is required for asexual conidiation, which is an important stage of the reproductive cycle, and for infection, in mold in nature [24], including in A. niger.The deletion of chsA in A. niger caused severe defects in aerial conidiation on PDA, SDAY, and CZA plates, which was in good agreement with those found in the disruption mutants of chsA (a class I CHS-encoding gene), chsB (a class II CHS-encoding gene), and chsC (a class III CHS-encoding gene) in A. fumigatus [7]; chsI-VII in M. acridum [6]; and chsA (a class II CHS-encoding gene) in A. nidulans [8] and the knockdown mutants of chs4 (a class III CHS-encoding gene) in P. chrysogenum [11].No significant differences were observed in the diameters of hyphal colonies among the WT and deletion mutants, which was similar to the findings in the disruption mutants of chsA, chsB, and chsC in A. fumigatus [7], and chsI, chsII, chsIV, and chsVI in M. acridum [6], but different from the findings in the disruption mutants of chsIII, chsV, and chsVII in M. acridum [6]; chsB (a class III CHS-encoding gene) in A. nidulans [8]; and the knockdown mutants of chs4 (a class III CHS-encoding gene) in P. chrysogenum [11].Accompanied by the reduction in the yields of conidia, much sparser and smaller conidiophores were observed in ΔchsA.Intriguingly, the transcriptional expression of upstream regulators (fluG, flbA, flbC, and flbE), velvet regulators (velB and vosA), and central regulators (brlA), which positively controlled conidiation in Aspergillus species [32], were significantly decreased in the ΔchsA culture grown on PDA plates in A. niger.These findings implied that chsA was positively involved in conidiation by

Discussion
In fungi, chitin synthases play important but differential roles in cell wall integrity, cellular growth and development, environmental adaptation, metabolism, and pathogenicity [1,6,7,10,11,14,[29][30][31].In this study, A. niger ChsA, a class II chitin synthase, was proven to be involved in conidiation, mycelial morphology, cell wall integrity, cellular response to oxidative stress, and the production of enzymes and organic acids, as discussed below.
Firstly, ChsA is required for asexual conidiation, which is an important stage of the reproductive cycle, and for infection, in mold in nature [24], including in A. niger.The deletion of chsA in A. niger caused severe defects in aerial conidiation on PDA, SDAY, and CZA plates, which was in good agreement with those found in the disruption mutants of chsA (a class I CHS-encoding gene), chsB (a class II CHS-encoding gene), and chsC (a class III CHS-encoding gene) in A. fumigatus [7]; chsI-VII in M. acridum [6]; and chsA (a class II CHS-encoding gene) in A. nidulans [8] and the knockdown mutants of chs4 (a class III CHS-encoding gene) in P. chrysogenum [11].No significant differences were observed in the diameters of hyphal colonies among the WT and deletion mutants, which was similar to the findings in the disruption mutants of chsA, chsB, and chsC in A. fumigatus [7], and chsI, chsII, chsIV, and chsVI in M. acridum [6], but different from the findings in the disruption mutants of chsIII, chsV, and chsVII in M. acridum [6]; chsB (a class III CHS-encoding gene) in A. nidulans [8]; and the knockdown mutants of chs4 (a class III CHS-encoding gene) in P. chrysogenum [11].Accompanied by the reduction in the yields of conidia, much sparser and smaller conidiophores were observed in ∆chsA.Intriguingly, the transcriptional expression of upstream regulators (fluG, flbA, flbC, and flbE), velvet regulators (velB and vosA), and central regulators (brlA), which positively controlled conidiation in Aspergillus species [32], were significantly decreased in the ∆chsA culture grown on PDA plates in A. niger.These findings implied that chsA was positively involved in conidiation by regulating the transcriptional expressions of some important regulators that are crucial for conidiation in A. niger.
Moreover, chitin synthases are important for chitin synthesis.The deletion of chsA led to the decrease in chitin content of hyphal cells in A. niger, which was in good agreement with the findings in ∆chsIV, as well as in ∆chsVI in M. acridum [6], ∆chs8 in F. graminearum [9], ∆chsC (a class III CHS-encoding gene) and ∆chsG (a class III CHS-encoding gene) in A. fumigatus [7], and the silencing mutant of chsC (a class III CHS-encoding gene) in A. niger [14]; however, it was opposite to the findings in ∆chsIV, ∆chsVI in M. acridum [6], ∆chsVII in P. graminearum [10], and ∆chsD (a class VII CHS-encoding gene) in A. fumigatus [7].It was alodifferent from the lack of significant changes in ∆chsI, ∆chsII, and ∆chsIII in M. acridum [6], ∆chsA (a class I CHS-encoding gene), ∆chsB (a class II CHS-encoding gene), ∆csmA (a class V CHS-encoding gene), ∆csmB (a class V CHS-encoding gene), and ∆chsF (a class IV CHS-encoding gene) in A. fumigatus [7].Chitin is a vital substance that constitutes the framework of the cell wall and maintains cell wall integrity and structure in fungi [33,34].Accompanied with the decrease in chitin content, the cell wall of the ∆chsA mutants became weakened and fragile, which was evidenced by more protoplasts being released in the ∆chsA mutants than in the WT.Moreover, ∆chsA mutants exhibited a thicker cell wall than the WT, which was similar to the thickened cell wall in the double deletion mutants of chsA (a class II CHS-encoding gene) and chsC (a class I CHS-encoding gene) in A. oryzae [8], but was contrary to the findings in ∆chsII, ∆chsIII, ∆chsIV, ∆chsV, and ∆chsV in M. acridum [6].Additionally, ∆chsA demonstrated that impaired cell wall integrity led to higher sensitivities to Congo red and SDS in A. niger, except for the altered cell wall fragility.The increased sensitivities to Congo red and SDS coincided with those in ∆chsV and ∆chsVI in M. acridum [6], ∆chs8 in F. graminearum [9], ∆chsVII in P. graminearum [10], ∆chsB (a class II CHS-encoding gene) in A. fumigatus [7], and ∆csmB (a class V CHS-encoding gene) and ∆csmA (a class V CHS-encoding gene) in A. fumigatus [7], but was in contrast to the increased resistance to Congo red in ∆chsA (a class I CHS-encoding gene), ∆chsC (a class III CHS-encoding gene) and ∆chsG (a class III CHS-encoding gene) in A. fumigatus [7], and ∆chsD (a class VII CHS-encoding gene) and ∆chsF (a class IV CHS-encoding gene) in A. fumigatus [7].
Apart from the defects in conidiation and cell wall integrity, the deletion of chsA led to the formation of looser and smaller pellets and a decrease in the number of septa in hyphal cells.The formation of looser and smaller pellets was opposite to the observation of more compact and/or bigger pellets in the silencing mutants of chsC (a class III CHS-encoding gene) in A. niger and chs4 (a class III CHS-encoding gene) in P. chrysogenum [11,14].The fewer septa in the hyphal cells differed from the lack of significant difference among the WT, ∆chsA (a class II CHS-encoding gene), and ∆chsC (a class I CHS-encoding gene) in A. nidulans, and among the WT, ∆chs1 (a class III CHS-encoding gene), and complementation mutants in Fusarium asiaticum [35,36].Generally, the spores of A. niger aggregate fast, subsequently germinate, and then form pellets during submerged fermentation [37].The deletion of chsA inhibited the aggregation of fungal conidia, which might be an explanation for the formation of looser and smaller pellets induced by the absence of chsA.In addition, changes in mycelial morphology are often associated with the production of metabolites during submerged fermentation.For example, dispersed mycelium is preferred for the production of gluconic acid and enzymes such as amylases, pectinases, fructofuranosidase, and proteases, whereas the pellet form is desirable for the production of citric acid and oxalate in A. niger [14,[38][39][40][41][42].Accompanied by the formation of more compact pellets, the silencing of chsC expression in A. niger improved the accumulation of citric acid during submerged fermentation [14].In this study, accompanied by the formation of looser and smaller pellets in ∆chsA, the deletion of chsA resulted in the decreased production of amylases, pectinases, cellulases, and malic acid, but increased citric acid production.The deletion of chsA would simultaneously destroy conidiation and cell wall integrity, which deteriorates the fermentation morphology and reduces the fermentation efficiency [43].For the decrease in the production of amylases, pectinases, cellulases, and malic acid, the negative effects of metabolic interference outweighed the positive effects of morphological optimization.In addition, both the transport of sugars and ammonia into the cell and the export of citrate ions from the cell are crucial to an understanding of the overproduction of citric acid [37].The cell wall plays an important role in material transport, so it was speculated that the increases in the fragility of the cell wall and the defects in cell wall integrity caused by the deletion of chsA might affect the transport of sugars, ammonia, citrate ions, and so on, which might lead to the increases in citric acid production in ∆chsA.
Furthermore, the cell wall is the first line of defense that protects microbes from environmental insults.Therefore, accompanied by the impaired cell wall in A. niger, cell tolerances to two typical oxidants (menadione and H 2 O 2 ) were decreased in ∆chsA.The decreased tolerances to menadione and/or H 2 O 2 were in accordance with the findings for ∆chsV and ∆chsVII in M. acridum [6], and ∆chsVII in P. graminearum [10], but differed from the lack of significant changes in ∆chsI, ∆chsII, ∆chsIII, ∆chsIV, and ∆chsVI in M. acridum [6], and ∆chs8 in F. graminearum [9].The CATs and SODs are the two key defense enzymes in the response to oxidative stress; therefore, their total activities are crucial to the response of A. niger to menadione and H 2 O 2 [31,44].The lowered activities of SODs and CATs coincided with the decreased tolerances caused by the deletion of chsA in A. niger.However, ∆chsA displayed a comparable sensitivity to NaCl and KCl as to the WT, which was similar to that in ∆chsI, ∆chsII, ∆chsIII, ∆chsIV, and ∆chsVI in M. acridum [6], and ∆chs8 in F. graminearum [9], but differed from the increased sensitivity to NaCl observed in ∆chs1 of Fusarium asiaticum [36].
In conclusion, chsA is required for mycelial morphology and cell wall integrity, including the chitin content, adaptive responses to cell wall-disturbing stresses, and the fragility and ultra-structure of the cell wall, and positively affects asexual conidiation, cellular responses to oxidative stresses, and the production of amylases, pectinases, cellulases, and malic acid, but negatively regulates the production of citric acid.

Figure 1 .
Figure 1.Structural features and phylogenetic analysis of ChsA in A. niger.(A) The conserved domains of ChsA.(B) Phylogenetic tree constructed for ChsA in A. niger and other fungal Class II CHSs in the NCBI database (accession codes given in parentheses) using a neighbor-joining method.Scale bar: branch length proportional to genetic distance.

Figure 1 .
Figure 1.Structural features and phylogenetic analysis of ChsA in A. niger.(A) The conserved domains of ChsA.(B) Phylogenetic tree constructed for ChsA in A. niger and other fungal Class II CHSs in the NCBI database (accession codes given in parentheses) using a neighbor-joining method.Scale bar: branch length proportional to genetic distance.

Figure 2 .
Figure 2. Roles of chsA in the asexual development of A. niger.(A) Images of fungal colonies after two-day growth on PDA, SDAY, and CZA plates at 30 °C.(B-D) Conidial yields during the period of incubation on PDA, SDAY, and CZA plates at 30 °C, respectively.(E) Relative transcript levels of conidiation-associated genes in ΔchsA versus WT.All cDNA samples were derived from one-dayold hyphal cells incubated on PDA plates and analyzed via qRT-PCR.The dashed line represents the transcriptional levels of genes in the WT.Asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD, p < 0.05).Error bars: SDs from three replicates.

Figure 2 .
Figure 2. Roles of chsA in the asexual development of A. niger.(A) Images of fungal colonies after two-day growth on PDA, SDAY, and CZA plates at 30 • C. (B-D) Conidial yields during the period of incubation on PDA, SDAY, and CZA plates at 30 • C, respectively.(E) Relative transcript levels of conidiation-associated genes in ∆chsA versus WT.All cDNA samples were derived from one-day-old hyphal cells incubated on PDA plates and analyzed via qRT-PCR.The dashed line represents the transcriptional levels of genes in the WT.Asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD, p < 0.05).Error bars: SDs from three replicates.

Figure 3 .
Figure 3. Images of conidial agglomeration (A), pellets morphology (A), and CFW staining (B) ( scale: 100 µm) observed under a microscope/fluorescence microscope when the suspension of conidia from the WT and ΔchsA were incubated at PDB with shaking at 30 °C for 48 h.The arrows showed the positions of the septa.

Figure 3 .
Figure 3. Images of conidial agglomeration (A), pellets morphology (A), and CFW staining (B) (scale: 100 µm) observed under a microscope/fluorescence microscope when the suspension of conidia from the WT and ∆chsA were incubated at PDB with shaking at 30 • C for 48 h.The arrows showed the positions of the septa.

Figure 4 .
Figure 4. Contributions of chsA to cell wall integrity of A. niger.(A) The content of total glucosamine based on the principle of the Morgan-Elson protocol.(B) Images of fungal colonies after two-day growth on PDA and PDA supplemented with Congo red (2 and 3 mg/mL) or SDS (0.03% and 0.04%) at 30 °C.(C) The number of protoplasts released from mycelial cells after 3.5 h treatment with cellwall-degrading enzymes.(D) TEM images for ultrathin sections of conidia (scales: 1.0 µm).Asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD, p < 0.05).Error bars: SDs from three replicates.

Figure 4 .
Figure 4. Contributions of chsA to cell wall integrity of A. niger.(A) The content of total glucosamine based on the principle of the Morgan-Elson protocol.(B) Images of fungal colonies after two-day growth on PDA and PDA supplemented with Congo red (2 and 3 mg/mL) or SDS (0.03% and 0.04%) at 30 • C. (C) The number of protoplasts released from mycelial cells after 3.5 h treatment with cellwall-degrading enzymes.(D) TEM images for ultrathin sections of conidia (scales: 1.0 µm).Asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD, p < 0.05).Error bars: SDs from three replicates.

Figure 5 .
Figure 5. Changes in the adaptive responses to oxidative and osmotic stresses and virulence caused by the deletion of chsA in A. niger.(A) Images of fungal colonies after two-day growth on PDA and PDA supplemented with KCl (1.2 M), NaCl (1.2 M), menadione (MND: 2 and 3 µM), or H2O2 (6 and 12 mM) at 30 °C.(B,C) The total activities of SODs (B) and CATs (C), respectively.(D) The infection ability on apple.Asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD, p < 0.05).Error bars: SDs from three replicates.

Figure 5 .
Figure 5. Changes in the adaptive responses to oxidative and osmotic stresses and virulence caused by the deletion of chsA in A. niger.(A) Images of fungal colonies after two-day growth on PDA and PDA supplemented with KCl (1.2 M), NaCl (1.2 M), menadione (MND: 2 and 3 µM), or H 2 O 2 (6 and 12 mM) at 30 • C. (B,C) The total activities of SODs (B) and CATs (C), respectively.(D) The infection ability on apple.Asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD, p < 0.05).Error bars: SDs from three replicates.

Figure 6 .
Figure 6.Effects of the deletion of chsA on the production of amylases (A), pectinases (B), cellulases (C), proteases (D), malic acid (E), and citric acid (F) when the suspension of conidia from the WT and ΔchsA were incubated at WBS with shaking at 30 °C for three days.Asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD, p < 0.05).Error bars: SDs from three replicates.

Figure 6 .
Figure 6.Effects of the deletion of chsA on the production of amylases (A), pectinases (B), cellulases (C), proteases (D), malic acid (E), and citric acid (F) when the suspension of conidia from the WT and ∆chsA were incubated at WBS with shaking at 30 • C for three days.Asterisked bars in each bar group differ significantly from those unmarked (Tukey's HSD, p < 0.05).Error bars: SDs from three replicates.