Glu1, a conserved GH30_3 β-(1,6)-glucanase requires its catalytic residues for full virulence in Ustilago maydis

Abstract

Plant-pathogenic fungi cause plant diseases and severe agricultural losses. The fungal cell wall is the first point of contact and defense barrier between fungus and host, maintaining cellular integrity and structural stability but also playing a dynamic role during infection. Through pathogenic development, the fungal cell wall must be continuously remodelled to accommodate morphological transitions and environmental challenges through diverse cell wall modifying enzymes necessary for successful infection. Here, we characterise Glu1, a putative endo-{beta}-(1,6)-glucanase (GH30) from the maize pathogen Ustilago maydis, conserved across Ustilaginales. Structural comparison with characterised GH30 crystal structures shows Glu1 most closely resembles GH30_3 {beta}-(1,6)-glucanases. Glu1 expression is induced during infection, localises to the plasma membrane in close association with the fungal cell wall and is detected in the secreted fraction. Deletion of glu1 reduces virulence, impairs plant colonization and tumour formation, and alters septation and cell morphology under saprophytic growth. Notably, genetic rescue with Glu1 catalytic-site variants largely restores saprophytic phenotypes but not virulence, suggesting a catalytic-dependent function of Glu1 specifically required for infection. Umag_04357, the other GH30 enzyme identified in U. maydis, reveals a conserved GH30 domain, including the catalytic residues, but is expressed at lower levels during infection and its deletion does not affect virulence. Together, our findings indicate that Glu1 is a conserved GH30 enzyme with a {beta}-(1,6)-glucanase-like pocket whose catalytic residues are required for full virulence. To our knowledge, it is the first GH30 enzyme linked to virulence in Basidiomycota and could be considered a potential target for smut disease control.