南方农业学报2026，Vol.57Issue(4)：1015-1026,12.DOI:10.3969/j.issn.2095-1191.2026.04.006
橡胶树白粉菌效应蛋白CSEP02974的糖苷水解酶活性测定及其免疫抑制功能分析
Determination of glycoside hydrolase activity and immunosup-pression function analysis of effector protein CSEP02974 in Erysiphe quercicola
摘要
Abstract
[Objective]This study aimed to determine the glycoside hydrolase activity of effector protein CSEP02974 in Erysiphe quercicola and analyze its immunosuppression function,providing theoretical basis for further investigation of functions and mechanisms of effector proteins in powdery mildew fungi.[Method]Phylogenetic analysis,domain se-quence alignment,and dbCAN 3 bioinformatics analysis were used to clarify the homology and conservation of the GH domain sequence of CSEP02974.CSEP02974 and its mutant proteins were expressed using a prokaryotic expression sys-tem.Activities of glycoside hydrolases(xylanase and β-1,3-glucanase)of the proteins were measured by in vitro enzyme activity assays to identify enzymatic active sites within the GH domain;CSEP02974 mutant proteins were expressed in Nicotiana benthamiana,and their suppression effects on hypersensitive death in leaves were assessed by leaf necrosis phe-notype observation and ion leakage rate determination;the CSEP02974 and its mutant proteins were added to CSEP02974-silenced fungal strains to clarify the relationship between the CSEP02974 activity and fungal pathogenicity.[Result]CSEP02974 belonged to the GH128 family,with 184 homologous proteins widely distributed in Leotiomycetes,Dothideomycetes,Eurotiomycetes,and Basidiomycota within Agaricomycetes,and highly conserved sites were present within the GH domain.CSEP02974 and its mutant proteins were obtained through prokaryotic expression and purification.Based on the GH domain sequence characteristics of CSEP02974,glutamic acid(Glu,E)within the GH domain at the N-terminal,middle,and C-terminal of the proteins to establish E1D(containing mutations E62D and E67D),E2D(con-taining mutations E86D,E88D,E100D,E143D,and E161D),and E3D(containing mutations E183D,E191D,E192D,E199D,E211D,and E239D).In vitro enzyme activity assays showed that CSEP02974 possessed xylanase activity,approximately 4.5 nmol/(min·mg),but lacked β-1,3-glucanase activity;E1D retained partial xylanase activity,approxi-mately 4.7 nmol/(min·mg),with no significant difference with CSEP02974(P>0.05,the same below),whereas E2D and E3D almost lost xylanase activity,and none of the mutant proteins showed β-1,3-glucanase activity.Expression of CSEP02974 in Nicotiana benthamiana effectively suppressed INF1-triggered hypersensitive necrosis,and the mutant pro-teins E2D and E3D still retained the plant immunosuppression function.CSEP02974-dsRNA treatment effectively silenced the gene encoding the effector protein and significantly reduced the disease area caused by Erysiphe quercicola;addition of CSEP02974 to CSEP02974-silenced strains restored the lesion area caused by the non-silenced strain(nuclease-free water treatment)from 5.00%to 29.74%.Addition of the mutant proteins E1D,E2D,and E3D also restored lesion areas to 33.67%,34.81%,and 30.38%respectively,whereas addition of the unrelated protein GST-His failed to restore lesion expansion.[Conclusion]CSEP02974 has xylanase catalytic activity,suggesting that it has the function of degrading plant cell wall,however,its roles in suppressing plant immunity and promoting pathogenicity do not depend on its xylanase activity.关键词
橡胶树白粉菌/效应蛋白/糖苷水解酶/植物免疫/致病性Key words
Erysiphe quercicola/effector protein/glycoside hydrolase/plant immunity/pathogenicity分类
农业科技引用本文复制引用
贺梦媛,彭洋,林春花,缪卫国,李潇..橡胶树白粉菌效应蛋白CSEP02974的糖苷水解酶活性测定及其免疫抑制功能分析[J].南方农业学报,2026,57(4):1015-1026,12.基金项目
国家自然科学基金项目(32402319) （32402319）
海南省科技人才创新项目(KJRC2023B14) National Natural Science Foundation of China(32402319) （KJRC2023B14）
Hainan Science and Technology Talent Innovation Project(KJRC2023B14) （KJRC2023B14）
