Supplementary MaterialsData_Sheet_1. covered most of -1,3-glucan GSK126 manufacturer synthesized by AgsA, although a small amount of -1,3-glucan was still present in the outer coating. We also constructed a strain with disruption of the gene, which encodes an intracellular -amylase that synthesizes -1,4-glucooligosaccharide like a primer for -1,3-glucan biosynthesis. With this strain, the hyphal pellets and maximum molecular mass of -1,3-glucan (94.5 1.4 kDa) were smaller than in the wild-type strain, and -1,3-glucan was still labeled with AGBD-GFP in the outermost coating. Overall, these results suggest that hyphal pellet formation depends on the molecular mass and spatial localization of -1,3-glucan as well as the amount of -1,3-glucan in the cell wall of have been fractionated into alkali-soluble (AS) and alkali-insoluble (AI) fractions (Fontaine et al., 2000). The AS portion GSK126 manufacturer consists of primarily -1,3-glucan with interconnecting -1,4-linkage, and some galactomannan (Bernard and Latg, 2001; Latg, 2010), and the AI portion is composed of chitin, -1,6-branched -1,3-glucan, and galactomannan (Fontaine et al., 2000; Bernard and Latg, 2001). The alkali solubility method has been applied to fractionate cell wall components of the model filamentous fungus (Yoshimi et al., 2013, 2015) and industrial fungi (Miyazawa et al., 2016; Zhang et al., 2017b); the components of polysaccharides in both fractions derived from the two fungi are similar to those derived from (Fontaine et al., 2000; Bernard and Latg, 2001). The part of -1,3-glucan in pathogenesis and hyphal adhesion has been reported in (Beauvais et al., 2005, 2013; Maubon et al., 2006; Fontaine et al., 2010; Henry et al., 2012; Yoshimi et al., 2013; Miyazawa et al., 2016; Zhang et al., 2017b). In and the pathogenic dimorphic candida drastically attenuates growth, and raises branching and cell lysis (Dichtl et GSK126 manufacturer al., 2015), which is similar to the phenotype of cells treated by caspofungin that is a -1,3-glucan synthase inhibitor. The family 1 chitin synthase mutants and of show reduced growth and modified mycelial morphotype (Muszkieta et al., 2014). In the family 2 chitin synthase mutant of offers three -1,3-glucan synthase genes (strain lacked -1,3-glucan and was less pathogenic than the parental strain. -1,3-Glucan of has a part in the aggregation of germinating conidia (Fontaine et al., 2010). The industrial fungus offers five -1,3-glucan synthase genes (and is up-regulated in the presence of cell wall stressCinducing compounds such as calcofluor white and caspofungin (Damveld et al., 2005). Among the three -1,3-glucan synthase genes of ((orthologous Mdk to (Grn et al., 2005) and (Choma et al., 2013). -Glucan from consists of two interconnected linear chains (subunits, 120 residues each) of 1 1,3-linked -glucose and some 1,4-linked -glucose residues at their reducing ends as spacers (Grn et al., 2005). Alkali-soluble glucan from your cell wall of consists of 25 subunits (200 residues each) of -1,3-glucan separated by a short spacer of 1 1,4-linked -glucose residues (Choma et al., 2013). offers two -1,3-glucan synthase genes, and gene prospects to the loss of -1,3-glucan; consequently, AgsB is required for -1,3-glucan biosynthesis under normal growth conditions (Yoshimi et al., 2013). In liquid culture, the disruptant offers fully dispersed hyphae, whereas the wild-type strain forms hyphal pellets (Yoshimi et al., 2013), suggesting that -1,3-glucan is definitely a hyphal aggregation element. The gene seems to be related to conidiation (He et al., 2014). However, the details of the function and the chemical structure of polysaccharides synthesized by AgsA and AgsB remain unclear. In is vital for -1,3-glucan synthesis, whereas overexpression of the GPI-anchored -amylase decreases the amount of cell wall -1,3-glucan (He et al., 2014). In the present study, we constructed the or strains, which overexpressed either or under the control of a constitutive promoter in the genetic background of or disruptants, respectively. The alkali-soluble glucan in the cell wall of these strains is composed of polysaccharides synthesized only by either AgsA or AgsB. In liquid culture, the irregular hyphal dispersion of the disruption strain was restored in the strain, which created hyphal pellets, suggesting that AgsA generates adhesive polysaccharides. The phenotypes of the hyphal pellets were obviously different between the and strains. We hypothesized that this difference is attributable to the difference in the chemical structure and/or the spatial localization in the cell wall of polysaccharides synthesized by AgsA or AgsB. In this study, we analyzed the chemical structure and localization of -1,3-glucan in the and strains. Materials and Methods Strains and Growth Press Strains are outlined in Supplementary Table S1. CzapekCDox (CD) medium was utilized for standard tradition (Fujioka et al., 2007). To fulfill the auxotrophic requirements of Gene The gene was disrupted by using the Cre/loxP marker recycling.