Abstract

Lysine 2-hydroxyisobutyrylation (K hib ) is a newly identified post-translational modification (PTM) that plays important roles in transcription and cell proliferation in eukaryotes. However, its function remains unknown in phytopathogenic fungi. Here, we performed a comprehensive assessment of K hib in the rice false smut fungus Ustilaginoidea virens , using Tandem Mass Tag (TMT)-based quantitative proteomics approach. A total of 3 426 K hib sites were identified in 977 proteins, suggesting that K hib is a common and complex PTM in U. virens . Our data demonstrated that the 2-hydroxyisobutyrylated proteins are involved in diverse biological processes. Network analysis of the modified proteins revealed a highly interconnected protein network that included many well-studied virulence factors. We confirmed that the Zn-binding reduced potassium dependency3-type histone deacetylase (UvRpd3) is a major enzyme that removes 2-hydroxyisobutyrylation and acetylation in U. virens . Notably, mutations of K hib sites in the mitogen-activated protein kinase (MAPK) UvSlt2 significantly reduced fungal virulence and decreased the enzymatic activity of UvSlt2. Molecular dynamics simulations demonstrated that 2-hydroxyisobutyrylation in UvSlt2 increased the hydrophobic solvent-accessible surface area and thereby affected binding between the UvSlt2 enzyme and its substrates. Our findings thus establish K hib as a major post-translational modification in U. virens and point to an important role for K hib in the virulence of this phytopathogenic fungus.