Analysis Of The Cell Wall Structure Of Basidiomycete Fugus By Fluorescence Labeling And Enzymatic Hydrolysis

Analysis of the cell wall structure of basidiomycete fugus by fluorescence labeling and enzymatic hydrolysis

Yuitsu otsuka1, Moriyuki Kawauchi1*, Kenya Tsuji1, Akira Yoshimi1, Chihiro Tanaka1,

 Takehito Nakazawa1, Shigekazu Yano2, Yoichi Honda1

1Graduate School of Agriculture, Kyoto University, Kitashirakawa-oiwakecho, Sakyo-ku, Kyoto 606-8502, Japan

2Graduate School of Sciences and Engineering, Yamagata University, Jonan, Yonezawa, Yamagata 992-8510, Japan

*Email: kawauchi.moriyuki.8c@kyoto-u.ac.jp

White-rot fungi belong to basidiomycete and have attracted attention because they can be cultivated using environmentally friendly renewable substrates such as wood and agricultural wastes. Their mycelia also have potential value as sustainable materials like vegan leather and plastic substitutes. The properties of fungal cell walls are deeply related to their usability as materials, but their structure, regulation, and synthesis mechanisms have not been characterized. In this study, we developed a polysaccharide staining method using three different fluorescent probes to visualize and analyze the cell wall structure in a model white-rot fungi Pleurotus ostreatus. α-1,3-glucan (AG), and β-glucan (BG) specific fluorescent probes as well as calcofluor-white for chitin staining were used for multicolor imaging of the cell wall. The outer layer of the cell wall was almost entirely covered with BG, indicating that BG is the main outer component. On the other hand, the hyphal tips and septum were rich in chitin. Furthermore, some AG-rich mycelia and AG particles on the outside of the mycelium were observed. A layer of AG appeared below the BG layer after β-1,3/1,6-glucanase treatment, and an inner layer of primarily chitin was observed close to the cell membrane after amylase treatment. Based on the results of enzymatic processing, P. ostreatus cell wall glucans are expected to contain mainly α-1,3- and β-1,3-linkages, with possible α-1,4-linkage, and β-1,6-/1,4-linkage branches. It is suggested that, although the major polysaccharides components are similar with those of ascomycetes, their distribution is completely different and unique in basidiomycete cell wall structure.