Comprehensive identification of lysine 2-hydroxyisobutyrylated proteins in Ustilaginoidea virens reveals the involvement of lysine 2-hydroxyisobutyrylation in fungal virulence

Lysine 2-hydroxyisobutyrylation(Khib) 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 Khibin the rice false smut fungus Ustilaginoidea virens, using Tandem Mass Tag(TMT)-based quantitative proteomics approach. A total of 3 426 Khibsites were identified in 977 proteins, sugg esting that Khibis a common and complex PTM in U. virens. Our data demonstrated that the2-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 Khibsites 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 Khibas a major post-translational modification in U. virens and point to an important role for Khibin the virulence of this phytopathogenic fungus.