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 Khib 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 Khibas a major post-translational modification in U. virens and point to an important role for Khibin the virulence of this phytopathogenic fungus.

Ustilaginoidea virens modulates lysine 2-hydroxyisobutyrylation in rice flowers during infection

The post-translational modification lysine 2-hydroxyisobutyrylation(K_(hib))plays an important role in gene transcription,metabolism,and enzymatic activity.K_(hib)sites have been identified in rice(Oryza sativa).However,the K_(hib)status of proteins in rice flowers during pathogen infection remains unclear.Here,we report a comprehensive identification of K_(hib)-modified proteins in rice flowers,and the changes in these proteins during infection with the fungal pathogen Ustilaginoidea virens.By using a tandem mass tag-based quantitative proteomics approach,we identified 2,891 K_(hib)sites on 964 proteins in rice flowers.Our data demonstrated that 2-hydroxyisobutyrylated proteins are involved in diverse biological processes.K_(hib)levels were substantially reduced upon infection with U.virens.Chromatin immunoprecipitation polymerase chain reaction(PCR)and reverse transcription quantitative PCR analyses revealed that histone K_(hib)is involved in the expression of disease-resistance genes.More importantly,most quantified sites on core histones H3 were downregulated upon U.virens infection.In addition,the histone deacetylases HDA705,HDA716,SRT1,and SRT2 are involved in the removal of K_(hib)marks in rice.HDA705 was further confirmed to negatively regulate rice disease resistance to pathogens U.virens,Magnaporthe oryzae,and Xanthomonas oryzae pv.oryzae(Xoo).Our data suggest that U.virens could modulate K_(hib)in rice flowers during infection.