S-palmitoylation of MAP kinase is essential for fungal virulence

acylation also called S-palmitoylation is an important reversible protein post-translational modification in organisms. However, its function remains unknown in pathogenic fungi. Here, we found treatment of rice false fungus Ustilaginoidea virens with S-palmitoylation inhibitor 2-BP resulted in a significant decrease in fungal virulence and growth rate. Deletion of the S-acyltransferase proteins from U. virens indicated that two S-acyltransferases UvPfa3 and UvPfa4 were required for fungal virulence. Comprehensive identification of S-palmitoylation sites and proteins in U. virens revealed a total of 4089 S-palmitoylation sites were identified within 2192 proteins and S-palmitoylation proteins were involved in diverse biological processes. Interestingly, S-palmitoylation proteins were significantly enriched in mitogen-activated protein kinase (MAPK) pathway and MAP kinase UvSlt2 was confirmed to be a S-palmitoylation protein which was palmitoylated by UvPfa4. Mutations of S-palmitoylation sites in UvSlt2 resulted in significantly reduced fungal virulence and decreased kinase enzymatic activity and phosphorylation level. Molecular dynamics simulations demonstrated mutation of S-palmitoylation sites in UvSlt2 caused decreased hydrophobic solvent-accessible surface area, and thereby affected binding between the UvSlt2 enzyme and substrates. Taken together, S-palmitoylation promotes U. virens virulence through palmitoylating MAP kinase UvSlt2 by palmitoyltransferase UvPfa4 and enhancing enzymatic activity and phosphorylation level of the kinase, thereby increasing hydrophobic solvent-accessible surface area and binding activity between the UvSlt2 enzyme and its substrates. Our studies provide a framework for dissecting the biological functions of S-palmitoylation, and reveal an important role for S-palmitoylation in regulating virulence of pathogen.

S-棕榈酰化（S-palmitoylation，又称酰化）是生物体内一类重要的可逆蛋白质翻译后修饰（protein post-translational modification）。然而，其在病原真菌（pathogenic fungi）中的功能仍未明晰。本研究发现，使用S-棕榈酰化抑制剂2-BP处理稻曲病菌（Ustilaginoidea virens）后，该真菌的致病力与生长速率均显著降低。通过敲除稻曲病菌的S-酰基转移酶（S-acyltransferase）蛋白，证实两种S-酰基转移酶UvPfa3与UvPfa4是真菌致病力所必需的因子。对稻曲病菌中的S-棕榈酰化位点与蛋白进行全面鉴定，共在2192个蛋白质中鉴定到4089个S-棕榈酰化位点，且S-棕榈酰化蛋白参与多种生物学过程。有趣的是，S-棕榈酰化蛋白显著富集于丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）通路，且证实MAP激酶UvSlt2为一类S-棕榈酰化蛋白，其棕榈酰化修饰由UvPfa4介导。对UvSlt2的S-棕榈酰化位点进行突变，可显著降低真菌致病力、激酶酶活性（kinase enzymatic activity）与磷酸化水平（phosphorylation level）。分子动力学模拟（molecular dynamics simulations）结果显示，UvSlt2的S-棕榈酰化位点突变会导致其疏水溶剂可及表面积（hydrophobic solvent-accessible surface area）降低，进而影响UvSlt2酶与其底物的结合能力。综上，S-棕榈酰化可通过棕榈酰转移酶（palmitoyltransferase）UvPfa4对MAP激酶UvSlt2进行棕榈酰化修饰，增强该激酶的酶活性与磷酸化水平，从而提升其疏水溶剂可及表面积与底物结合活性，最终促进稻曲病菌的致病力。本研究为解析S-棕榈酰化的生物学功能提供了系统性研究框架，并揭示了S-棕榈酰化在调控病原真菌致病力中的重要作用。