Functional analysis of the α-1,3-glucan synthase genes agsA and agsB in Aspergillus nidulans: agsB is the major α-1,3-glucan synthase in this fungus.

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and (13)C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species

Cell wall susceptibility of the control (CNT), <i>agsB</i> disruption (DagsB), <i>agsA agsB</i> double disruption (DagsA-DagsB), CagsB, and CagsB with <i>agsA</i> disruption (CagsB-DagsA) strains to Lysing Enzymes.

<p>(<b>A</b>) Susceptibility of mycelia cultured in CD liquid medium (<i>agsB-</i>repressing conditions). Mycelia cultured in CD medium for 24 h (30 mg fresh weight) were digested in reaction buffer (10 mM phosphate buffer, pH 6.0) containing 10 mg/mL Lysing Enzymes. After 1, 2, and 4 h of incubation at 30°C, the number of protoplasts in each sample was determined using a hemocytometer. Error bars represent the standard deviations (<i>n</i> = 3). All values of the mutants differed significantly from that of the control strain. (<b>B</b>) Susceptibility of the control and CagsB strains cultured in CDTF liquid medium (<i>agsB-</i>inducing conditions). Mycelia cultured in CDTF medium for 24 h (30 mg fresh weight) were digested, and the number of protoplasts in each sample was determined according to the method described above. None of the differences were statistically significant. (<b>C</b>) Effect of combined treatment with α-1,3-glucanase and Lysing Enzymes on protoplast formation from fungal mycelia. Mycelia cultured in CD medium for 24 h were digested in reaction buffer containing both Lysing Enzymes and α-1,3-glucanase. The number of protoplasts generated in each sample was determined according to the method described above. None of the differences were statistically significant.</p

Sensitivity of the control (CNT), <i>agsB</i> disruption (DagsB), <i>agsA agsB</i> double disruption (DagsA-DagsB), CagsB, and CagsB with <i>agsA</i> disruption (CagsB-DagsA) strains to Congo Red (CR).

<p>(<b>A</b>) Growth rate after 4 days of growth on CD medium at the indicated concentration of added CR. Error bars represent the standard deviations (<i>n</i> = 3). *, significantly different from the control (<i>P</i><0.01). (<b>B</b>) Adsorption of CR to the hyphae of each strain. Mycelia cultured in CD for 24 h (500 mg fresh weight) were suspended in 50 mL of CR solution (40 µg/mL) and rotated (160 rpm) for 1 h at room temperature. After the reaction, the mycelia were removed by filtration, and the absorbance of the supernatant was measured at 500 nm. Absorbance levels indicate the levels of CR remaining in the supernatants (i.e., not adsorbed to the hyphae). Error bars represent the standard error of the mean calculated for three replicates (*<i>P</i><0.05).</p

Expression of cell wall–related genes in the control (CNT) and CagsB strains.

<p>Strains were grown in CD liquid medium (<i>agsB</i>-repressing conditions) or CDTF liquid medium (<i>agsB</i>-inducing conditions) at 37°C. Levels of transcription of the indicated genes were determined by means of quantitative RT-PCR of total RNA using the gene-specific primers reported previously by Fujioka et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054893#pone.0054893-Fujioka1" target="_blank">[10]</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054893#pone.0054893.s010" target="_blank">Table S1</a>). Each value represents the ratio of expression relative to the histone H2B gene in each strain. Bars represent the standard error of the mean calculated for at least three replicates (*<i>P</i><0.05, **<i>P</i><0.01).</p

Hyphal morphology of the control and <i>agsB</i> disruption strains grown in CD liquid medium for 12 h at 37°C.

<p>(<b>A</b>) Hyphal pellets of the control strain, showing hyphal aggregation (dark areas). Scale bar = 200 µm. (<b>B</b>) Hyphal tip morphology of the control strain. High magnification of the hyphal image in the white frame in panel A. Scale bar = 50 µm. (<b>C</b>) Dispersed hyphae of the <i>agsB</i> disruption strain (DagsB). Scale bar = 200 µm. (<b>D</b>) Hyphal tip morphology of the DagsB strain. High magnification of the hyphal image in the white frame in panel A. Scale bar = 50 µm.</p

Expression of cell wall–related genes in the control (CNT) and <i>agsB</i> disruption (DagsB) strains.

<p>Strains were grown in CD liquid medium at 37°C. Levels of transcription of the indicated genes were determined by means of quantitative RT-PCR of total RNA using the gene-specific primers reported previously by Fujioka et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054893#pone.0054893-Fujioka1" target="_blank">[10]</a> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054893#pone.0054893.s010" target="_blank">Table S1</a>). Each value represents the ratio of expression relative to the histone H2B gene in each strain. Error bars represent the standard error of the mean calculated for three replicates (*<i>P</i><0.05, **<i>P</i><0.01).</p