Stress responsive glycosylphosphatidylinositol-anchored protein SsGSP1 contributes to <i>Sclerotinia sclerotiorum</i> virulence
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Fungal cell wall acts as a defense barrier, shielding the cell from varying environmental stresses. Cell wall proteins, such as glycosylphosphatidylinositol (GPI)-anchored proteins, are involved in swift and appropriate responses to minor environmental changes in fungi. However, the roles of these proteins in the pathogenic <i>Sclerotinia sclerotiorum</i> remain largely unexplored. Here, we identified a novel GPI-anchored protein in <i>S. sclerotiorum</i>, SsGSP1, comprising a Kre9_KNH domain. <i>SsGSP1</i> was upregulated during infection, and the loss-of-function mutants of <i>SsGSP1</i> exhibited the compromised cell wall integrity and reduced β-glucan content. During inoculation on <i>Arabidopsis thaliana</i>, <i>Nicotiana benthamiana</i>, and <i>Brassica napus</i>, the <i>SsGSP1</i>-deletion strains demonstrated the decreased virulence. The transgenic <i>A</i>. <i>thaliana</i> line carrying the sRNA targeting <i>SsGSP1</i> enhanced resistance to <i>S. sclerotiorum</i> via Host-Induced Gene Silencing (HIGS). The <i>SsGSP1</i>-deficient strains displayed the heightened sensitivity to various stresses, including osmotic pressure, oxidative stress, and heat shock. The yeast two-hybrid and BiFC assays confirmed that SsGSP1 interacted with the key stress-related proteins catalase SsCat2, heat shock protein Sshsp60, and ABC transporter SsBMR1. Accordingly, transcriptome analysis revealed that the disruption of <i>SsGSP1</i> downregulated the expression of genes involved in oxidative stress response, heat shock response, and chemical agent resistance. These results collectively delineate the intricate role of GPI-anchored protein SsGSP1 in β-glucan, cell wall integrity, and virulence and may act as a potential surface sensor to elicit signal transduction in response to environmental stresses in <i>S. sclerotiorum</i>.
真菌细胞壁作为防御屏障，可保护细胞免受各类环境胁迫的侵害。细胞壁蛋白，例如糖基磷脂酰肌醇（glycosylphosphatidylinositol, GPI）锚定蛋白，参与真菌对细微环境变化的快速且恰当的响应过程。然而，此类蛋白在致病真菌核盘菌（*Sclerotinia sclerotiorum*）中的功能仍未得到充分探索。本研究在核盘菌（*S. sclerotiorum*）中鉴定到一种新型GPI锚定蛋白SsGSP1，其包含一个Kre9_KNH结构域。SsGSP1在侵染过程中表达上调；SsGSP1功能缺失突变体的细胞壁完整性受损，且β-葡聚糖含量降低。在接种拟南芥（*Arabidopsis thaliana*）、本氏烟（*Nicotiana benthamiana*）与油菜（*Brassica napus*）时，SsGSP1敲除菌株的致病力显著下降。表达靶向SsGSP1的sRNA的转基因拟南芥品系，可通过寄主诱导的基因沉默（Host-Induced Gene Silencing, HIGS）增强对核盘菌的抗性。SsGSP1缺陷菌株对多种胁迫的敏感性升高，包括渗透压胁迫、氧化胁迫与热激胁迫。酵母双杂交（yeast two-hybrid）与双分子荧光互补（BiFC）实验证实，SsGSP1可与关键胁迫相关蛋白——过氧化氢酶SsCat2、热激蛋白Sshsp60以及ABC转运蛋白SsBMR1发生互作。相应地，转录组分析显示，SsGSP1的敲除会下调氧化胁迫响应、热激响应以及化学药剂抗性相关基因的表达。综上，本研究结果阐明了GPI锚定蛋白SsGSP1在β-葡聚糖代谢、细胞壁完整性维持以及致病力调控中的复杂功能，其可能作为一种潜在的表面感受器，介导核盘菌响应环境胁迫的信号转导过程。
