A Glycosylphosphatidylinositol-Anchored α-Amylase Encoded by amyD Contributes to a Decrease in the Molecular Mass of Cell Wall α-1,3-Glucan in Aspergillus nidulans

α-1,3-Glucan is one of the main polysaccharides in the cell wall of Aspergillus nidulans. We previously revealed that it plays a role in hyphal aggregation in liquid culture, and that its molecular mass (MM) in an agsA-overexpressing (agsAOE) strain was larger than that in an agsB-overexpressing (agsBOE) strain. The mechanism that regulates its MM is poorly understood. Although the gene amyD, which encodes glycosylphosphatidylinositol (GPI)-anchored α-amylase (AmyD), is involved in the biosynthesis of α-1,3-glucan in A. nidulans, how it regulates this biosynthesis remains unclear. Here we constructed strains with disrupted amyD (ΔamyD) or overexpressed amyD (amyDOE) in the genetic background of the ABPU1 (wild-type), agsAOE, or agsBOE strain, and characterized the chemical structure of α-1,3-glucans in the cell wall of each strain, focusing on their MM. The MM of α-1,3-glucan from the agsBOE amyDOE strain was smaller than that in the parental agsBOE strain. In addition, the MM of α-1,3-glucan from the agsAOE ΔamyD strain was greater than that in the agsAOE strain. These results suggest that AmyD is involved in decreasing the MM of α-1,3-glucan. We also found that the C-terminal GPI-anchoring region is important for these functions.

Outside of A. fumigatus, A. nidulans agsB and its orthologs are clustered with two α-amylase-encoding genes (amyD and amyG in A. nidulans) (He et al., 2014;Yoshimi et al., 2017;Miyazawa et al., 2020).The amyG gene encodes an intracellular α-amylase and is crucial for α-1,3-glucan synthesis (He et al., 2014).The amyD gene in A. nidulans encodes glycosylphosphatidylinositol (GPI)-anchored α-amylase.He et al. (2014) reported that α-1,3-glucan contents increased by 50% in an amyD-disrupted ( amyD) strain and halved in an amyD-overexpressing (actA(p)-amyD) strain, suggesting that amyD has a repressive effect on α-1,3-glucan synthesis.In addition, He et al. (2017) analyzed the chronological changes of α-1,3-glucan contents under liquid culture conditions.Whereas, the amount of α-1,3-glucan in strains that overexpressed the α-1,3-glucanase-encoding gene (mutA or agnB) was decreased after 20 h from inoculation, the amount of α-1,3-glucan in the cell wall of the amyD OE strain was half that of the wild-type strain from the initial stage of cultivation (He et al., 2017).He et al. (2017) suggested that AmyD decreased the amount of α-1,3-glucan in the cell wall by a mechanism independent of the effect of α-1,3-glucanase.The enzymatic characteristics of A. niger AgtA, which is encoded by an ortholog of A. nidulans amyD, have been reported (Van Der Kaaij et al., 2007).Although AgtA in A. niger barely hydrolyzed α-1,3-glucan, it had relatively high transglycosylation activity on donor substrates with maltooligosaccharides (Van Der Kaaij et al., 2007).Overall, AmyD seems to indirectly decrease the amount of α-1,3-glucan in the cell wall, but the detailed mechanism is still unknown.Here, in a study of the function of amyD in α-1,3glucan biosynthesis in A. nidulans, we constructed strains with overexpression or disruption of amyD in the genetic backgrounds of the wild-type, agsA OE , and agsB OE .We performed several chemical analyses of α-1,3-glucan derived from the strains, looking in particular at its MM, and examined the role of amyD in controlling the MM of α-1,3-glucan in the cell wall.

Strains and Growth Media
Strains are listed in Table 1.Czapek-Dox (CD) medium was used as the standard culture, as described previously (Fujioka et al., 2007;Miyazawa et al., 2018).

Construction of the agsA-and agsB-Overexpressing Strains
We newly constructed agsA OE and agsB OE strains for this study.To generate agsA OE , pAPyT-agsA plasmids (Miyazawa et al., 2018) were digested with NotI and transformed into a disrupted agsB ( agsB) strain (Supplementary Figure 1A).Correct integration of agsA overexpression cassettes was confirmed by PCR (Supplementary Figure 1B).To generate agsB OE , the disrupted agsA ( agsA) strain was first generated using the Cre/loxP marker recycling system (Zhang et al., 2017a).The pAPG-cre/DagsA plasmid (Miyazawa et al., 2018) was digested with EcoRI and transformed into the ABPU1 (argB + ) strain.Candidate strains were selected on CD medium without uridine and uracil, and then cultured on CD medium with uridine and uracil and 1% xylose to induce Cre expression (Supplementary Figure 1C).Strains that required uridine and uracil were isolated, and then replacement of the agsA gene was confirmed by PCR (Supplementary Figure 1D).The pAPyT-agsB plasmid was digested with NotI and transformed into the agsA strain (Supplementary Figure 1E).Correct integration of agsB overexpression cassettes was confirmed by PCR (Supplementary Figure 1B).

Construction of the amyD OE Strain
The amyD OE strain was constructed by replacing the native promoter with the constitutive tef1 promoter.The sequences of the primers are listed in Supplementary Table 1.To generate amyD OE , the plasmid pAPT-amyD was constructed (Supplementary Figure 2A).The 5 ′ -non-coding region (amplicon 1) and the coding region (amplicon 2) of amyD were amplified from A. nidulans ABPU1 genomic DNA.The pyrG marker (amplicon 3) was amplified from the pAPGcre/DagsA plasmid.The tef1 promoter (amplicon 4) was amplified from the pAPyT-agsB plasmid.The four amplicons and a SacI-digested pUC19 vector were fused using an In-Fusion HD Cloning Kit (Clontech Laboratories, Inc., Mountain View, CA, USA).The resulting plasmid was digested with SacI, and transformed into the ABPU1 (argB + ), agsA OE , and agsB OE strains (Supplementary Figure 2B).Correct integration of the cassette was confirmed by PCR (Supplementary Figure 2C).

Disruption of the amyD Gene
In the first round of PCR, gene fragments containing the 5 ′ -noncoding region (amplicon 1) and the coding region (amplicon 2) of amyD were amplified from ABPU1 genomic DNA, and the pyrG gene (amplicon 3) was amplified from A. oryzae genomic DNA (Supplementary Figure 2D).The three resulting fragments were gel-purified and fused into a disruption cassette in the second round of PCR.The resulting PCR product was gel-purified and transformed into the ABPU1 (argB + ), agsA OE , and agsB OE strains (Supplementary Figure 2E).Replacement of the amyD gene was confirmed by PCR (Supplementary Figure 2F).

RNA Extraction and Quantitative Real-Time PCR
Mycelial cells cultured in CD liquid medium for 24 h were collected, and total RNA was extracted from the cells by using Sepasol-RNA I Super G (Nakalai Tesque, Kyoto, Japan) in accordance with the manufacturer's instruction.The total RNA (2.5 µg) was reverse-transcribed by using a SuperScript IV VILO Master Mix with ezDNase Enzyme (Invitrogen, Carlsbad, CA, United States).Quantitative real-time PCR was performed with a Mx3000P (Agilent Technologies, Santa Clara, CA, United States) with SYBR Green detection.For reaction mixture preparation, Thunderbird Next SYBR qPCR Mix (Toyobo Co., Ltd., Osaka, Japan) was used.Primers used for quantitative PCR are listed in Supplementary Table 1.An equivalent amount of cDNA, obtained from reverse transcription reactions using an equivalent amount of total RNA, was applied to each reaction mixture.
The gene encoding histone H2B was used as a normalization reference (an internal control) for determining the target gene expression ratios.

Delipidization and Fractionation of Mycelial Cells
Cell walls were fractionated as previously described with some modification (Miyazawa et al., 2018).Mycelia cultured for 24 h in CD medium were collected by filtering through Miracloth (Merck Millipore, Darmstadt, Germany), washed with water, and freeze-dried.The mycelia were then pulverized in a MM400 bench-top mixer mill (Retch, Haan, Germany).The powder (1 g) was suspended in 25 mL of chloroform-methanol (3:1 vol/vol) and stirred at room temperature for 12 h to remove the total polar lipid content of the mycelial cells.The mixture was centrifuged (10,000× g, 10 min).The residue was suspended in chloroformmethanol, and the delipidizing procedure was repeated.Then the de-polar lipid residue was suspended in 40 mL of 0.1 M Na phosphate buffer (pH 7.0), and cell-wall components were fractionated by hot-water and alkali treatments, as described previously (Miyazawa et al., 2018).Hot-water-soluble, alkalisoluble, and alkali-insoluble fractions were obtained from this fractionation, and the alkali-soluble fraction was further separated into a fraction soluble in water at neutral pH (AS1) and an insoluble fraction (AS2).The monosaccharide composition of AS2 fractions was quantified according to Miyazawa et al. (2018).
To obtain mycelia cultured for 16 h, conidia (final conc.5.0 × 10 5 /mL) were inoculated into 200 mL CD medium and rotated at 160 rpm at 37 • C. The mycelia were collected and fractionated as described above.

C NMR Analysis
The AS2 fraction of each strain (50 mg) was suspended in 1 mL of 1 M NaOH/D 2 O and dissolved by vortexing.One drop of DMSOd 6 (deuterated dimethyl sulfoxide) was then added to each fraction and the solutions were centrifuged (3,000 × g, 5 min) to remove insoluble debris. 13C NMR spectra of the supernatants were obtained using a JNM-ECX400P spectrometer (JEOL, Tokyo, Japan) at 400 MHz at 35 • C (72,000 scans).Chemical shifts were recorded relative to the resonance of DMSO-d 6 .

Determination of the Average Molecular Mass of Alkali-Soluble Glucan
The MM of alkali-soluble glucan was determined by gel permeation chromatography (GPC) according to the methods of Puanglek et al. (2016), with some modification.A GPC-101 system (Showa Denko Co. Ltd., Tokyo, Japan) with an ERC-3125S degasser (Showa Denko) and an RI-71S refractive index detector (Showa Denko) was used for the measurement.It was fitted with a GPC KD-G 4A guard column (Showa Denko) and a GPC KD-805 column (8.0 × 300 mm; Showa Denko).The eluent was 20 mM LiCl in N, N-dimethylacetamide (DMAc), and the flow rate was 0.6 mL/min at 40 • C. The detector was normalized with polystyrene standards (SM-105; Showa Denko).With SmartChrom software (Jasco, Tokyo, Japan), the GPC profile was divided into virtual time slices (n i ) with the height of each virtual slice from the base line (H i ) corresponding to a certain MM (M i ) obtained by calibrating the column.From these values, the number-average MM (M n ) and weight-average MM (M w ) were calculated as:

Smith Degradation
Smith degradation of the alkali-soluble glucan was performed as described (Miyazawa et al., 2018).In brief, the AS2 fraction (20 mg) was suspended in 0.1 M acetate buffer (pH 3.9), oxidized with sodium periodate, reduced with sodium borohydride, hydrolyzed with trifluoroacetic acid, and freeze-dried.These procedures resulted in selective hydrolyzing of the 1,4-linked glucose residues, which contain vicinal hydroxyl groups, but not the 1,3-glucose residues in alkali-soluble glucan.The Smithdegraded sample was dissolved in DMAc containing LiCl for GPC analysis.

Western Blotting
Culture broth in CD liquid medium incubated for 24 h was filtered through Miracloth.Proteins in the supernatant were precipitated with trichloroacetic acid, separated by SDS-PAGE, and then transferred onto a polyvinylidene difluoride membrane.
The membrane was blocked with the polyvinylidene difluoride blocking reagent for Can Get Signal (Toyobo).Antibodies of rabbit IgG against AmyD were developed with synthesized peptides (NH 2 -C+SGERAGELDVPMSK-COOH) (Eurofins Genomics, Tokyo, Japan) and used as the primary antibody, diluted with Can Get Signal (Toyobo).Antibody binding was visualized using a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Pierce Biotechnology, Rockford, IL, USA) and an ImmunoStar LD chemiluminescent substrate (Fujifilm Wako Pure Chemical Corp., Osaka, Japan).

Assay for Glucosyltransferase Activity
Supernatant obtained from CD liquid medium cultured for 24 h was concentrated 167 times and buffer-exchanged to 10 mM Tris•HCl (pH 8.0) in Nanosep Centrifugal Devices 10K (Pall, Port Washington, NY, USA).A 20-µL mixture containing 1 mM para-nitrophenyl (pNP)-α-maltopentaoside and 1 mM acarbose (Fujifilm Wako Pure Chemical Corp.), and 5 µL of the concentrated culture supernatant in 50 mM acetic acid/sodium acetate buffer (pH 5.5) was incubated for 20 min at 40 • C. Samples (3 µL) were withdrawn from the reaction mixture and immediately inactivated by adding 40 µL of methanol.pNPα-Maltopentaoside was prepared by following the method of Usui and Murata (1988).Then, 157 µL of water was added to each sample solution, which was analyzed by HPLC with a Jasco Intelligent System liquid chromatograph (Jasco).The bound material was eluted with 20% methanol at a flow rate of 1.0 mL/min at 40 • C. The elution profiles were detected at 300 nm with a Unison UK C-18 column (4.6 × 250 mm, Imtakt, Kyoto, Japan).

Characterization of Strains With Disrupted or Overexpressed amyD
We constructed amyD OE and amyD strains by introducing the amyD cassettes for overexpression and disruption into the wildtype, agsA OE , and agsB OE strains (Supplementary Figure 2).The expression level of amyD in each strain was quantified in hyphal cells.Whereas, each disrupted strain ( amyD, agsA OE amyD, and agsB OE amyD) showed scarce amyD expression, each overexpressing strain (amyD OE , agsA OE amyD OE , and agsB OE amyD OE ) showed significantly higher amyD expression than their parental strain (Figure 1).
There was no significant difference in radial growth among the strains grown on agar plates for 5 days (Supplementary Figure 4).In liquid culture, the wild-type and amyD strains formed tightly aggregated hyphal pellets; however, the hyphae of the amyD OE strain were almost fully dispersed (Figure 2).He et al. reported that the phenotype of their amyD OE strain resembles that of the agsB strain in A. nidulans (He et al., 2014), which is consistent with our results (Figure 2).In agreement with our previous results (Miyazawa et al., 2018), the agsA OE and agsB OE strains formed, respectively, loosely and tightly aggregated pellets (Figure 2).Disruption of amyD did not affect the phenotypes of the agsA OE and  agsB OE strains (Figure 2).Also, overexpression of amyD scarcely affected the phenotypes of the agsA OE and agsB OE strains (Figure 2).

Overexpression of amyD Resulted in a Decrease in Cell-Wall Alkali-Soluble Glucan
Cell-wall components of each strain were fractionated by a hot water-alkali treatment method, each fraction was weighed, and the monosaccharide composition of the AS2 fraction was quantified.The amount of glucose in the AS2 fraction was significantly lower in the amyD OE strain than in the wild-type strain (Figure 3; P < 0.05).That in the amyD strain was similar to that in the wild-type strain (Figure 3).Those in the agsA OE amyD OE and agsA OE amyD strains, which were constructed from the parental strain agsA OE , were almost the same (Figure 3).It was significantly lower in the agsB OE amyD OE strain than in the agsB OE and agsB OE amyD strains (Figure 3; P < 0.05).These results indicate that AmyD acts to decrease the amount of alkalisoluble glucan in the wild-type and agsB OE strains, but not in the agsA OE strain, even when amyD is overexpressed.

Overexpression of the amyD Gene Decreases the Molecular Mass of Alkali-Soluble Glucan
By 13 C NMR analysis, the primary component in the AS2 fraction of the wild-type, amyD OE , and amyD strains was found to be α-1,3-glucan, suggesting that amyD did not affect the primary components of alkali-soluble glucan (Supplementary Figure 5).To reveal whether the MM of alkali-soluble glucan was affected by disruption or overexpression of amyD, we determined the MM of alkali-soluble glucan in each strain by GPC analysis.Polystyrene (MM, 13,900-3,850,000) was used as a standard molecule to calibrate the column for size exclusion analysis.Although M w is used to assess the physical properties of a  polymer, the calculation of M w favors molecules with a larger MM.Since M n is the average of the MM values of the individual macromolecules, here we use M n as the MM of alkali-soluble glucan.The M n of the alkali-soluble glucan was 1,260,000 ± 270,000 in the agsA OE strain and 312,000 ± 5,000 in agsB OE strain (Figures 4A,B; Table 2), consistent with our previous results (Miyazawa et al., 2018).Although the M n of alkali-soluble glucan in the agsA OE amyD OE strain (1,110,000 ± 110,000) was similar to that in the parental (agsA OE ) strain, that of agsA OE amyD was significantly greater (2,250,000 ± 130,000) than that of agsA OE (Figure 4A; Table 2; P < 0.05).In addition, the M n of agsB OE amyD OE (140,000 ± 8,000) was significantly less than that of the parental (agsB OE ) strain (Figure 4B; Table 2; P < 0.05).The M n of alkali-soluble glucan in agsB OE amyD (358,000 ± 19,000) was similar to that in agsB OE (Figure 4B; Table 2).Lastly, the M n of alkali-soluble glucan in the wildtype (2,280,000 ± 320,000) and amyD (2,390,000 ± 400,000) was larger than that in agsB OE (312,000 ± 5,000; Figure 4C; Table 2).The amyD OE strain had a primary peak at around 17 min (M 1 n , 32,900 ± 300) and a secondary peak at 11 min (M 2 n , 2,210,000 ± 700,000).These results suggest that AmyD degraded the alkali-soluble glucan eluted around 11 min to Values are mean ± standard deviation of three replicates.produce alkali-soluble glucan with a smaller MM (Figure 4C; Table 2).
Although the alkali-soluble glucan in the wild-type strains was synthesized mainly by AgsB, its MM was larger than that in the agsB OE strain (Table 2).Additionally, when agsB was overexpressed, the amount of α-1,3-glucan was three times that in the wild-type (Figure 3).We supposed that some unknown glycosyl modification enzymes may contribute to the increase in MM of α-1,3-glucan in the wild-type, and that because the agsB OE strain produces more α-1,3glucan, there is little modification by the unknown enzymes.Therefore, we determined the MM of alkali-soluble glucan extracted from 16-h cultured mycelia, which should be less affected by the modification enzyme than the 24-h cultured mycelia (He et al., 2017).Unexpectedly, the M n of the alkali-soluble glucan in the mycelia cultured for 16 h was 1,980,000 ± 320,000, which was similar to that in the mycelia cultured for 24 h (1,930,000 ± 280,000; Supplementary Figure 6; Supplementary Table 2).We then evaluated the MM of alkali-soluble glucan in A4, which is the Glasgow wild-type of A. nidulans (Table 1), and found it had M n = 2,224,000 ± 390,000 (Supplementary Table 3), which is similar to that in the wild-type strain.
To validate whether the degree of polymerization of α-1,3-glucan subunits in the alkali-soluble glucan was altered when the MM was changed by amyD disruption or overexpression, we applied Smith degradation to the alkali-soluble glucan from each strain to selectively cleave 1,4-linked glucan, and then determined the MM by GPC.One subunit of α-1,3-glucan in the alkalisoluble glucan is composed of ≈200 glucose residues (Choma et al., 2013;Miyazawa et al., 2018).The Smithdegraded alkali-soluble glucan in each strain had almost the same MM, equivalent to 300-400 glucose residues (Supplementary Figure 7; Supplementary Table 4), which suggests that AmyD activity does not decrease the degree of polymerization of the glucose residues in each α-1,3-glucan subunit.

Spatial Localization of α-1,3-Glucan in the Cell Wall Is Not Affected by amyD Disruption or Overexpression
We previously revealed that spatial localization of α-1,3-glucan in the cell wall changes according to its MM (Miyazawa et al., 2018); α-1,3-glucans in agsB OE cells are localized in the outer layer in the cell wall, whereas most of those in the agsA OE cells are masked by a β-1,3-glucan layer.In this study, disruption or overexpression of amyD altered the MM of alkali-soluble glucan (Figure 4; Table 2); therefore, we analyzed whether this alteration affected the spatial localization of α-1,3-glucan in the cell wall.In agreement with previous results (Miyazawa et al., 2018), the α-1,3-glucans with AGBD-GFP labeling showed clearly in the wild-type and agsB OE cells, but only weakly in agsA OE cells (Figure 5).The amyD and amyD OE cells were also labeled with AGBD-GFP (Figure 5); fluorescent intensity in amyD OE was relatively low, which might be caused by a decrease in the amount of alkali-soluble glucan in the cell wall of amyD OE cells.The labeling with AGBD-GFP in agsA OE amyD OE and agsA OE amyD cells was weak, as was that in the cells of the parental agsA OE strain (Figure 5).The agsB OE amyD cells were clearly labeled with AGBD-GFP, as in the parental agsB OE (Figure 5).The AGBD-GFP labeling was slightly weaker in agsB OE amyD OE than in agsB OE , which might be attributable to a decrease in the amount of α-1,3-glucan.After treatment with β-1,3-glucanase, α-1,3-glucans of the hyphal cells in agsA OE , agsA OE amyD OE , and agsA OE amyD cells were clearly labeled with AGBD-GFP (Supplementary Figure 8), suggesting that these strains have α-1,3-glucan in the inner layer of the cell wall in their hyphal cells.Taken together, these findings indicate that disruption or overexpression of amyD gene scarcely affected the spatial localization of α-1,3-glucan in the cell wall.
The GPI Anchor Is Essential for the Effect of AmyD on Both the Amount and Molecular Mass of Alkali-Soluble Glucan AmyD is thought to contain a GPI anchor at the C-terminal region.Fungal GPI anchor-type proteins are transferred from the plasma membrane to the cell wall by the activity of the GH76 family (Vogt et al., 2020).We speculated that localization in the cell wall would be essential for AmyD to reach the substrate, alkali-soluble glucan, so we constructed overexpression strains of amyD with and without the GPI-anchor site.Because we noticed that overexpression of amyD alters the phenotype or the alkali-soluble glucan, we used amyD and agsB OE amyD strains as hosts for the amyD OE strains.The hyphae of amyD formed pellets in shake-flask culture (Figure 6).Those of amyD-amyD OE were dispersed, as in amyD OE (Figure 6).Those of amyD-amyD OE ( GPI) formed pellets, although the form was slightly different from that in the parental strain (Figure 6).Those of agsB OE amyD, agsB OE amyD-amyD OE , and agsB OE amyD-amyD OE ( GPI) formed similar pellets (Figure 6).Although the amyD-amyD OE hyphae had less AS2-Glc (1.13% ± 0.21%) than amyD (5.68% ± 0.25%), the amount was restored in amyD-amyD OE ( GPI) hyphae (5.17% ± 0.46%; Figure 7A).These results suggest that the GPI anchor of AmyD has an important negative effect on α-1,3-glucan biosynthesis.The hyphae of agsB OE amyD-amyD OE had marginally less AS2-Glc (16.2% ± 0.6%) than those of agsB OE amyD (17.6% ± 0.3%) and agsB OE amyD-amyD OE ( GPI) (16.7% ± 0.5%; Figure 7B).We then evaluated the MM of alkalisoluble glucan in the cells of agsB OE amyD, agsB OE amyD-amyD OE , and agsB OE amyD-amyD OE ( GPI).The M n of the alkali-soluble glucan in agsB OE amyD-amyD OE cells (174,000 ± 8,000) was smaller than that in agsB OE amyD (270,000 ± 8,000; Figure 7C; Table 3; P < 0.05).The M n of alkali-soluble glucan in agsB OE amyD-amyD OE ( GPI) cells (349,000 ± 42,000) was similar to that in agsB OE amyD (Figure 7C; Table 3).These results suggest that the GPI anchor of AmyD is also important for regulating the MM of alkali-soluble glucan.
Western blotting showed that secretion of AmyD in the culture supernatant could be detected only in the amyD-amyD OE ( GPI) strain (Supplementary Figure 9A).Because AgtA in A. niger has relatively high transglycosylation activity on donor substrates with maltooligosaccharides (Van Der Kaaij et al., 2007), we evaluated the α-amylase activity in concentrated culture supernatants with pNP-α-maltopentaoside.Although various products possibly produced by coexisting α-glucosidase were detected in amyD and both amyD-overexpressing strains, pNP-α-maltooctaoside (probably a transglycolylation product of AmyD) was detected only in the amyD-amyD OE ( GPI) strain (data not shown).A histidine residue in region I, which is a highly conserved region of α-amylases, is substituted to the asparagine residue in AmyD and proteins encoded by orthologs of amyD (Van Der Kaaij et al., 2007).Substitution  Values are mean ± standard deviation of three replicates.
of the histidine residues in α-amylase increases the inhibitory constant (Ki) of a representative α-glucosidase inhibitor, acarbose (Svensson, 1994).Therefore, we evaluated the αamylase activity in concentrated culture supernatants in the presence of acarbose.As expected, the hydrolysis product of pNP-α-maltopentaoside was hardly detected in amyD (Supplementary Figure 9B).pNP-α-maltoside and pNP-αmaltooctaoside were clearly detected in the supernatant of amyD-amyD OE ( GPI), but scarcely in that of amyD-amyD OE (Supplementary Figure 9B).These results suggest that although enzymatically active AmyD is secreted into the culture supernatant, it cannot decrease the MM of α-1,3-glucan, leading us to suppose that active AmyD needs to be localized on the plasma membrane or in the cell wall space to regulate the MM of α-1,3-glucan.

DISCUSSION
Although the GPI-anchored α-amylase AmyD is known to be involved in the biosynthesis of α-1,3-glucan in A. nidulans (He et al., 2014(He et al., , 2017)), the detailed mechanism remains unclear.Here, we looked at strains with disrupted or overexpressed amyD to analyze how AmyD affects the chemical properties of alkalisoluble glucan.The results reveal that overexpression of amyD not only decreased the MM of α-1,3-glucan, but also decreased the amount of α-1,3-glucan in the cell wall.The GPI anchor of AmyD was essential in both actions.
Overexpression of amyD affected the amount and MM of α-1,3-glucan in the wild-type and agsB OE strains, but not in the agsA OE strain (Figures 3, 4; Table 2).We previously reported that the MM of α-1,3-glucan controls where α-1,3-glucan is localized in the cell wall of A. nidulans; namely, that the α-1,3-glucan with a larger MM that is synthesized by AgsA is localized in the inner layer of the cell wall, and the smaller one that is synthesized by AgsB is localized in the outer layer (Miyazawa et al., 2018).To explain the effect of AmyD on the amount and MM of α-1,3-glucan, we formed the following two hypotheses from the results of this study.(1) Given that fungal GPI-anchored proteins are transferred from the plasma membrane to the cell wall (Orlean, 2012;Gow et al., 2017), our findings suggest that AmyD decreased the MM of α-1,3glucan localized at the outer layer of the cell wall.The increased MM of alkali-soluble glucan in the agsA OE amyD strain can be explained by its GPC elution profiles, which suggest that the MM of the polysaccharides was broadly distributed (Figure 4A); in other words, agsA OE amyD had mainly α-1,3-glucan with larger MM [>623,000 (M p of alkali-soluble glucan from agsB OE ), 97.5%], but also had a small amount of α-1,3-glucan with small MM (<623,000, 2.5%).We speculate that this small amount of α-1,3-glucan with a smaller MM may be localized in the outer layer of the cell wall of agsA OE , where it is accessible to AmyD, which results in the small amount of α-1,3-glucan with a smaller MM.(2) Generally, as the degree of polymerization increases, the solubility of polysaccharides in water decreases (Guo et al., 2017).We have previously explained that after the biosynthesis of α-1,3glucan on the plasma membrane, sugar chains are released to the outside of the membrane, where they are gradually insolubilized and immobilized to become a part of the cell wall (Miyazawa et al., 2018).Because α-1,3-glucan molecules with a larger MM might be more quickly insolubilized than those with a smaller MM, they are consequently localized in the inner layer of the cell wall, whereas α-1,3-glucan with a smaller MM might more likely be distributed toward the outer layer of the cell wall.α-1,3-Glucan synthesized with smaller MM in the agsB OE strain, which takes a relatively long time to become insoluble, seems to be more catalytically accessible by AmyD than α-1,3-glucan synthesized with larger MM in the agsA OE strain.To understand the relationship between the spatial localization of AmyD and α-1,3-glucan in the cell wall, immunoelectron microscopic and glycochemical analyses are necessary and are our future work.AmyD of A. nidulans is considered to be a GPI-anchored protein (De Groot et al., 2009;He et al., 2014).It is wellknown that many fungal GPI-anchored proteins are related to remodeling of the cell wall (Samalova et al., 2020).Proteins in the "defective in filamentous growth" (DFG) family recognize the GPI core glycan and then transfer to the β-1,3-or β-1,6-glucan (Muszkieta et al., 2019;Vogt et al., 2020), which allows GPI-anchored proteins to react with their substrates in the cell wall.Although there is no direct evidence that DFG family proteins contribute to transglycosylation in Aspergillus species, their role in cell-wall integrity in A. fumigatus was recently reported (Li et al., 2018;Muszkieta et al., 2019), which implies that DFG family proteins are important for transferring the GPI-core glycan to β-glucan in Aspergillus species.To reveal the importance of the GPI anchor in the function of AmyD, we evaluated the MM and amount of α-1,3-glucan in amyD-overexpressing strains with or without the GPI-anchoring site.Interestingly, decreases in the MM and the amount of α-1,3-glucan were not observed when the C-terminal GPIanchoring site was deleted (Figure 7; Table 3); amyD-amyD OE ( GPI) formed slightly altered pellets (Figure 6), suggesting that AmyD expressed without its GPI anchor has only partial functions.Furthermore, western blotting detected the secretion of AmyD in the culture supernatant from the amyD-amyD OE ( GPI) strain (Supplementary Figure 9A).AgtA in A. niger has relatively high glucosyltransferase activity toward donor substrates with maltooligosaccharides (Van Der Kaaij et al., 2007).In a separate study, we expressed and purified A. oryzae AgtA (homologous to A. nidulans AmyD) in Pichia pastoris, and the purified A. oryzae AgtA showed α-amylase (hydrolysis and transferase) activity toward pNP-α-maltopentaoside (Koizumi et al., unpublished).Therefore, we evaluated the α-amylase activity in culture supernatants with pNP-α-maltopentaoside.Whereas, the hydrolysis product of pNP-α-maltopentaoside was hardly detected in amyD (Supplementary Figure 9B), the supernatant from amyD-amyD OE ( GPI) produced pNP-αmaltoside and pNP-α-maltooctaoside at an early stage of the reaction (Supplementary Figure 9B).These results suggest that enzymatically active AmyD that is secreted into the culture supernatant cannot decrease the amount and MM of α-1,3glucan.Taken together, the results show that expression of AmyD with a GPI anchor is important for reaching the substrate, α-1,3glucan, in the space of the cell wall.
Cell-wall polysaccharides are thought to be synthesized on the plasma membrane after the secretory vesicles containing polysaccharide synthases have been exported to the hyphal tip (Riquelme, 2013).On the basis of our previous findings (Miyazawa et al., 2020), we hypothesize the process of alkalisoluble glucan biosynthesis of A. nidulans to be as follows: (1) the intracellular domain of α-1,3-glucan synthase polymerizes 1,3-linked α-glucan chains from UDP-glucose as a substrate from the primers, which are maltooligosaccharides produced by intracellular α-amylase AmyG; (2) the elongated glucan chain is exported to the extracellular space through the multitransmembrane domain of α-1,3-glucan synthase; (3) the extracellular domain of α-1,3-glucan connects several chains of the elongated glucan to form mature alkali-soluble glucan.The mechanism underlying the distribution of mature alkali-soluble glucan to the cell-wall network is still unknown.However, the water solubility of newly synthesized glucan might be related to the spatial distribution of α-1,3-glucan in the cell wall, because localization of α-1,3-glucan varies according to the difference in MM (Miyazawa et al., 2018).Aspergillus niger AgtA (encoded by an ortholog of A. nidulans amyD) scarcely hydrolyzes α-1,3glucan and shows weak hydrolytic activity to starch (Van Der Kaaij et al., 2007).Therefore, decrease of the MM of alkali-soluble glucan in the amyD OE strain could be caused by hydrolysis of the primer/spacer residues (1,4-linked α-glucan) rather than of the 1,3-linked α-glucan region.The mechanism underlying the decrease in the amount of α-1,3-glucan by AmyD is also unknown.He et al. (2017) reported that AmyD seems to directly repress α-1,3-glucan synthesis.We suspect that AmyD with a GPI anchor on the plasma membrane binds to the spacer residues of a glucan chain that is being just synthesized by α-1,3-glucan synthase, and competitively inhibits transglycosylation by the extracellular domain of α-1,3-glucan synthase to decrease the amount of alkali-soluble glucan in the cell wall.
The M n of the alkali-soluble glucan from the wild-type strain was larger than that from the agsB OE , although the alkali-soluble glucan from both strains seemed to be synthesized mainly by AgsB (Figure 4; Table 2).The M n of the alkali-soluble glucan in the 16-h-cultured mycelia from the wild-type was similar to that from the 24-h-cultured mycelia (Supplementary Figure 6; Supplementary Table 2).α-1,3-Glucan was clearly labeled with AGBD-GFP in the wild-type strain (Figure 5).These results suggest that α-1,3-glucan was located in the outer layer of the cell wall in the wild-type strain, consistent with the localization of α-1,3-glucan synthesized by AgsB.These results imply the existence of some factor that increases the MM of α-1,3-glucan.We surmise that once a matured α-1,3glucan molecule synthesized by AgsB is localized in the outer layer of the cell wall, macromolecules are formed by interconnecting α-1,3-glucan or connecting α-1,3-glucan to other polysaccharides, resulting in a chemically stable complex.Although the difference was not significant, the MM of Smithdegraded alkali-soluble glucan in the wild-type strain was slightly higher (Supplementary Table 4) and its GPC profile had a broader distribution (Supplementary Figure 7) than those in the agsA OE and agsB OE strains, implying the existence of non-Smith-degradable glycosidic bonds (i.e., β-1,3-glycosidic bond) the alkali-soluble fraction in the wild-type strain.It is well-known that β-glucan, chitin, and galactomannan are continuously modified by hydrolase or glycosyltransferase in the cell wall (Aimanianda et al., 2017;Henry et al., 2019;Muszkieta et al., 2019).However, an enzyme that modifies α-1,3-glucan has not been reported.The recent report by Kang et al. (2018) on the cell wall architecture of A. fumigatus suggested the presence of a covalent bond of α-1,3-glucan to β-1,3-and β-1,4-glucan.The report by Chakraborty et al. (2021) on cell wall organization by whole-cell NMR showed that α-1,3-glucan fractionated into both alkali-soluble andinsoluble fractions for the rigid and mobile portions.An enzyme that has a role in modifying α-1,3-glucan to allow its transition into the different portions needs to be identified in the near future.
Here, we revealed that AmyD in A. nidulans decreased the MM of the alkali-soluble glucan composed mainly of α-1,3glucan in the cell wall and also the amount of alkali-soluble glucan.However, a complete picture of the biosynthesis of α-1,3-glucan has yet to be described, because the substrates or proteins associated with α-1,3-glucan synthesis have not been directly demonstrated.To unveil the true nature of the biosynthesis, further biochemical analysis of the α-1,3-glucan synthase is essential.

FIGURE 1 |
FIGURE 1 | Transcript levels of amyD determined by quantitative PCR.Gene-specific primers are listed in Supplementary Table1.Error bars represent the SEM calculated from three replicates.*Significant differences by Tukey's test (P < 0.05); n.s., not significant.

FIGURE 2 |
FIGURE 2 | Growth characteristics of amyD OE and amyD strains in liquid culture.Upper images, cultures in Erlenmeyer flasks; lower images, representative hyphal pellets of each strain under a stereomicroscope.Scale intervals are 1 mm.

FIGURE 3 |
FIGURE 3 | Amount of glucose in AS2 fractions.Conidia (5.0 × 10 5 /mL) of each strain were inoculated into CD medium and rotated at 160 rpm at 37 • C for 24 h.Values show glucose content of the AS2 fraction as a percentage of the total cell-wall weight.Error bars represent SEM calculated from three replicates.*Significant difference by Tukey's test (P < 0.05); n.s., not significant.

FIGURE 4 |
FIGURE 4| GPC elution profile of the AS2 fraction from the series of (A) agsA OE strains, (B) agsB OE strains, and (C) wild-type The AS2 fraction from 24-h-cultured mycelia of each strain was dissolved in 20 mM LiCl/DMAc.The elution profile was monitored by a refractive index detector.Molecular mass (MM) of the glucan peaks was determined from a calibration curve of polystyrene (PS) standards ( ).M w , weight-average MM; M n , number-average MM.

FIGURE 6 |
FIGURE 6 | Growth characteristics of amyD-amyD OE strains in liquid culture.Upper images, cultures in Erlenmeyer flasks; lower images, representative hyphal pellets of each strain under a stereomicroscope.Scale intervals are 1 mm.

FIGURE 7 |
FIGURE 7 | (A,B) Amounts of glucose and (C) GPC elution profiles of the AS2 fraction in amyD-amyD OE strains.(A,B) Conidia (5.0 × 10 5 /mL) of each strain were inoculated into CD medium and rotated at 160 rpm at 37 • C for 24 h.Values show glucose content of AS2 fraction as a percentage of the total cell-wall weight.Error bars represent SEM calculated from three replicates.*Significant difference by Tukey's test (*P < 0.05); n.s., not significant.(C) The AS2 fraction from 24-h-cultured mycelia of each strain was dissolved in 20 mM LiCl/DMAc.The elution profile was monitored by a refractive index detector.Molecular mass (MM) of the glucan peaks was determined from a calibration curve of polystyrene (PS) standards ( ).M w , weight-average MM; M n , number-average MM.

TABLE 1 |
Strains used in this study.
a Fungal Genetic Stock Center, USA.

TABLE 2 |
Molecular mass of alkali-soluble glucan in the cell wall.
a AS2, insoluble components after dialysis of the alkali-soluble fraction.b Peak molecular mass.c Weight-average molecular mass.d Number-average molecular mass.

TABLE 3 |
Molecular mass of alkali-soluble glucan in the cell wall of amyD-amyD OE strains.
b Peak molecular mass.c Weight-average molecular mass.d Number-average molecular mass.