生物性逆境（biotic stress）是影響全球糧食安全的重要因素之一，其來源包括細菌、真菌、線蟲與昆蟲等病原生物。玉米黑穗病菌（Ustilago maydis）是一種營養寄生型（biotrophic）真菌病原菌，會引起玉米黑穗病。為了促進感染，真菌在宿主定殖過程中會分泌效應蛋白（effector proteins），以抑制植物免疫反應並操控宿主細胞功能。然而，由於許多真菌效應蛋白缺乏明顯的序列相似性，因此難以進行功能註解，使得以蛋白質結構為基礎的分析成為解析其分子機制的重要方法。 本研究利用蛋白質結構預測來探討兩個先前已被鑑定為毒力因子的 U. maydis 效應蛋白 Um5027 與 Um5819 的功能。針對 Um5819，結構與序列分析鑑定出三個精胺酸殘基（arginine residues），其對於葉綠體在質膜上的錨定作用是必要的。BiFC 分析顯示，Um58193RA 突變株仍可與葉綠體上的 CHUP1 互作，但無法將葉綠體錨定於質膜，也失去抑制 BAX 誘導細胞死亡的能力，顯示這些精胺酸殘基對宿主操控功能具有關鍵作用。 針對 Um5027，其蛋白質結構預測顯示與 CBM49 具有高度相似性，並含有四個保守的色胺酸殘基（tryptophan residues），暗示其可能具有醣類結合功能。結構導向的突變分析顯示，將第 53 與第 176 位色胺酸突變為丙胺酸後，雖不影響蛋白分泌，但會喪失毒力功能，顯示這些殘基對效應蛋白功能至關重要。此外，Um5027 能抑制枯草桿菌（Bacillus subtilis）的生長，顯示其可能在宿主定殖過程中的微生物競爭中扮演角色。 總結而言，本研究顯示蛋白質結構預測可作為解析真菌效應蛋白功能的強大工具，有助於揭示 U. maydis 毒力機制以及宿主–病原交互作用的分子基礎。;Biotic stress caused by pathogens， such as bacteria， fungi， nematodes， and insects， severely threatens global food security. The biotrophic fungus Ustilago maydis causes smut disease in maize by secreting effector proteins to suppress host immunity and manipulate cellular processes. As many of these effectors lack functional annotation due to low sequence similarity， structure-based analysis offers a powerful tool to uncover their molecular mechanisms. In this study， we employed structural predictions to investigate two previously identified U. maydis effectors， Um5027 and Um5819. Structural and functional analyses of Um5819 identified three arginine (Arg) residues at positions R74， R76， and R80 essential for anchoring chloroplasts to the plasma membrane. While the triple-Arg mutant Um58193RA retained its interaction with the chloroplast protein CHUP1， it failed to anchor chloroplasts to the plasma membrane and could no longer suppress BAX-induced cell death， indicating the functional importance of these Arg residues in host manipulation. For Um5027， structural modeling revealed high similarity to carbohydrate-binding module family 49 (CBM49) and identified four conserved tryptophan residues. Structure-guided mutagenesis showed that substituting tryptophans at positions 53 and 176 with leucine abolished virulence without affecting protein secretion. Furthermore， Um5027 exhibited antibacterial activity against Bacillus subtilis， suggesting a role in microbial competition during host colonization. Collectively， this work highlights how predicted protein structures provide a powerful framework for deciphering the molecular mechanisms of uncharacterized fungal effectors during host colonization.
