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

Abstract

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 13C-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.

Mitogen-activated protein kinases MpkA and MpkB independently affect micafungin sensitivity in Aspergillus nidulans

“…24) The sequences of all primers used in this study are listed in Table S1. To disrupt a gene in A. nidulans, we replaced it with A. oryzae argB or A. nidulans pyrG as a selectable marker.…”

“…In addition, the pyrG-alcA(p) cassette, 24) which contains the A. nidulans pyrG marker and the alcA promoter (originally derived from A. nidulans FGSC A89 strain), was amplified by PCR using the primers pyrG-F and PalcA-R (Table S1). These three PCR-amplified fragments were used as substrates for the second-round PCR fusion (Fig.…”

“…24) Primer sets for quantifying cell wall-related gene expression 6,24) are listed in Table S1. The gene for histone H2B was used as a normalization reference (internal control) for target gene expression ratios.…”

“…24) Mycelia cultivated in liquid CD medium at 37°C for 24 h were collected and freeze-dried. According to their solubility in alkali, the freeze-dried mycelia were fractionated into four fractions: hot-water-soluble (HW); alkali-soluble 1 (AS1), which contained the water-soluble components after dialysis of the alkali-soluble fraction; alkali-soluble 2 (AS2), which contained the water-insoluble components after dialysis of the alkali-soluble fraction; and alkaliinsoluble (AI).…”

“…The procedure for quantification of the carbohydrate composition of the cell wall fractions has been described previously. 24) Each freeze-dried cell wall fraction (10 mg dry weight) was hydrolyzed in sulfuric acid, and the carbohydrate composition was determined using high-performance chromatography with a pulsed electrochemical detector and an anion-exchange column (Carbo PAC PA-1, 4 × 25 mm; Dionex, Sunnyvale, CA, USA).…”