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

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.

Automated summary

The study identified lysine 2-hydroxyisobutyrylation (K-hib) as a common and complex post-translational modification in the phytopathogenic fungus Ustilaginoidea virens, involved in diverse biological processes and connected to virulence factors in a protein network analysis. The enzyme UvRpd3 was confirmed to remove 2-hydroxyisobutyrylation and acetylation, while mutations in K-hib sites of the MAPK UvSlt2 led to reduced virulence and enzymatic activity in the fungus. Molecular dynamics simulations showed that 2-hydroxyisobutyrylation in UvSlt2 affected its binding to substrates by increasing hydrophobic solvent-accessible surface area.