AUTHOR=Wang Yanxin , Li Ding , Li Zhoukun , Cui Zhongli , Ye Xianfeng 

TITLE=Functional analysis of a novel endo-β-1,6-glucanase MoGlu16 and its application in detecting cell wall β-1,6-glucan of Magnaporthe oryzae

JOURNAL=Frontiers in Microbiology

VOLUME=15

YEAR=2024

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2024.1429065

DOI=10.3389/fmicb.2024.1429065

ISSN=1664-302X

ABSTRACT=<p>As an essential component of the fungal cell wall, β-1,6-glucan has an important role in the growth and development of fungi, but its distribution has not been investigated in <italic>Magnaporthe oryzae</italic>. Here, a novel β-1,6-glucanase from <italic>M. oryzae</italic>, <italic>Mo</italic>Glu16, was cloned and expressed in <italic>Pichia pastoris</italic>. The enzyme was highly active on pustulan, with a specific activity of 219.0 U/mg at pH 5.0 and 50°C, and showed great selectivity for continuous β-1,6-glycosidic bonding polysaccharides. Based on this, β-1,6-glucan was selectively visualized in the vegetative hyphae, conidia and bud tubes of <italic>M. oryzae</italic> using a hydrolytically inactive GFP-tagged <italic>Mo</italic>Glu16 with point mutations at the catalytic position (His-<italic>Mo</italic>Glu16<sup>E236A</sup>-Gfp). The spore germination and appressorium formation were significantly inhibited after incubation of 10<sup>5</sup>/ml conidia with 0.03 μg/μl <italic>Mo</italic>Glu16. Mycelia treated with <italic>Mo</italic>Glu16 produced reactive oxygen species and triggered the cell wall integrity pathway, increasing the expression levels of genes involved in cell wall polysaccharide synthesis. These results revealed that <italic>Mo</italic>Glu16 participated in the remodeling of cell wall in <italic>M. oryzae</italic>, laying a foundation for the analysis of cell wall structure.</p>