AUTHOR=Miyazawa Ken , Yoshimi Akira , Kasahara Shin , Sugahara Asumi , Koizumi Ami , Yano Shigekazu , Kimura Satoshi , Iwata Tadahisa , Sano Motoaki , Abe Keietsu 

TITLE=Molecular Mass and Localization of α-1,3-Glucan in Cell Wall Control the Degree of Hyphal Aggregation in Liquid Culture of Aspergillus nidulans

JOURNAL=Frontiers in Microbiology

VOLUME=9

YEAR=2018

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.02623

DOI=10.3389/fmicb.2018.02623

ISSN=1664-302X

ABSTRACT=<p>α-1,3-Glucan is one of the main polysaccharides in the cell wall of filamentous fungi. <italic>Aspergillus nidulans</italic> has two α-1,3-glucan synthase genes, <italic>agsA</italic> and <italic>agsB</italic>. We previously revealed that AgsB is a major α-1,3-glucan synthase in vegetative hyphae, but the function of AgsA remained unknown because of its low expression level and lack of phenotypic alteration upon gene disruption. To clarify the role of α-1,3-glucan in hyphal aggregation, we constructed strains overexpressing <italic>agsA</italic> (<italic>agsA<sup>OE</sup></italic>) or <italic>agsB</italic> (<italic>agsB<sup>OE</sup></italic>), in which the other α-1,3-glucan synthase gene was disrupted. In liquid culture, the wild-type and <italic>agsB<sup>OE</sup></italic> strains formed tightly aggregated hyphal pellets, whereas <italic>agsA<sup>OE</sup></italic> hyphae aggregated weakly. We analyzed the chemical properties of cell wall α-1,3-glucan from the <italic>agsA<sup>OE</sup></italic> and <italic>agsB<sup>OE</sup></italic> strains. The peak molecular mass of α-1,3-glucan from the <italic>agsA<sup>OE</sup></italic> strain (1,480 ± 80 kDa) was much larger than that from the wild type (147 ± 52 kDa) and <italic>agsB<sup>OE</sup></italic> (372 ± 47 kDa); however, the peak molecular mass of repeating subunits in α-1,3-glucan was almost the same (after Smith degradation: <italic>agsA<sup>OE</sup></italic>, 41.6 ± 5.8 kDa; <italic>agsB<sup>OE</sup></italic>, 38.3 ± 3.0 kDa). We also analyzed localization of α-1,3-glucan in the cell wall of the two strains by fluorescent labeling with α-1,3-glucan-binding domain<italic>–</italic>fused GFP (AGBD-GFP). α-1,3-Glucan of the <italic>agsB<sup>OE</sup></italic> cells was clearly located in the outermost layer, whereas weak labeling was detected in the <italic>agsA<sup>OE</sup></italic> cells. However, the <italic>agsA<sup>OE</sup></italic> cells treated with β-1,3-glucanase were clearly labeled with AGBD-GFP. These observations suggest that β-1,3-glucan covered most of α-1,3-glucan synthesized by AgsA, although a small amount of α-1,3-glucan was still present in the outer layer. We also constructed a strain with disruption of the <italic>amyG</italic> gene, which encodes an intracellular α-amylase that synthesizes α-1,4-glucooligosaccharide as a primer for α-1,3-glucan biosynthesis. In this strain, the hyphal pellets and peak molecular mass of α-1,3-glucan (94.5 ± 1.4 kDa) were smaller than in the wild-type strain, and α-1,3-glucan was still labeled with AGBD-GFP in the outermost layer. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of α-1,3-glucan as well as the amount of α-1,3-glucan in the cell wall of <italic>A. nidulans</italic>.</p>