Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery m...Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of Hv WRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of Hv WRKY2 requires an intact WRKY domain and an upstream sequence of~75 amino acids,and the Hv WRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of Hv WRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of Hv WRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.Hv CEBiP positively regulates resistance against Bgh in barley.Our findings suggest that Hv WRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that Hv CEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.展开更多
植物转录因子是植物体内调节基因表达的重要蛋白,参与多种生物学功能的调控。研究植物转录因子调控靶基因的常用方法为染色质免疫共沉淀法(chromatin immunoprecipitation,ChIP),但抗体特异性、遗传材料构建的耗时性等因素却大大限制了C...植物转录因子是植物体内调节基因表达的重要蛋白,参与多种生物学功能的调控。研究植物转录因子调控靶基因的常用方法为染色质免疫共沉淀法(chromatin immunoprecipitation,ChIP),但抗体特异性、遗传材料构建的耗时性等因素却大大限制了ChIP技术的应用范围和效果。本文通过对水稻原生质体瞬时转化、甲醛固定、免疫沉淀核酸的超声波破碎等条件的优化,建立了基于水稻原生质体的染色质免疫共沉淀技术体系(chromatin immunoprecipitation system based on rice protoplasts,ChIP-RP);并通过本技术体系验证了水稻转录因子OsNF-YA4蛋白对靶基因序列的富集作用。本技术体系将减少制备特异性抗体或构建稳定遗传材料的局限,有利于水稻转录因子直接调控靶基因的快速筛选和验证,推动水稻转录因子调控功能的分子机制解析。展开更多
基金supported by National Key Research and Development Program of China(2018YFD1000703,2018YFD1000700)Strategic Priority Research Program of the Chinese Academy of Sciences(XDB11020400)+3 种基金National Program on Research and Development of Transgenic Plants(2016ZX08009-003-001)Startup Fund for Advanced Talents of Lushan Botanical GardenChinese Academy of Science(2020ZWZX03 and 2020ZWZX05)the“Double Hundred and Double Thousand”Talent Project of Jiujiang City(jjsbsq2020026)。
文摘Plants use a sophisticated immune system to perceive pathogen infection and activate immune responses in a tightly controlled manner.In barley,Hv WRKY2 acts as a repressor in barley disease resistance to the powdery mildew fungus,Blumeria graminis f.sp.hordei(Bgh).However,the molecular features of Hv WRKY2 in its DNA-binding and repressor functions,as well as its target genes,are uncharacterized.We show that the W-box binding of Hv WRKY2 requires an intact WRKY domain and an upstream sequence of~75 amino acids,and the Hv WRKY2 W-box binding activity is linked to its repressor function in disease resistance.Chromatin immunoprecipitation(ChIP)-seq analysis identified HvCEBiP,a putative chitin receptor gene,as a target gene of Hv WRKY2 in overexpressing transgenic barley plants.ChIP-qPCR and Electrophoretic Mobility Shift Assay(EMSA)verified the direct binding of Hv WRKY2 to a W-boxcontaining sequence in the HvCEBiP promoter.Hv CEBiP positively regulates resistance against Bgh in barley.Our findings suggest that Hv WRKY2 represses barley basal immunity by directly targeting pathogen-associated molecular pattern(PAMP)recognition receptor genes,suggesting that Hv CEBiP and likely chitin signaling function in barley PAMP-triggered immune responses to Bgh infection.
文摘植物转录因子是植物体内调节基因表达的重要蛋白,参与多种生物学功能的调控。研究植物转录因子调控靶基因的常用方法为染色质免疫共沉淀法(chromatin immunoprecipitation,ChIP),但抗体特异性、遗传材料构建的耗时性等因素却大大限制了ChIP技术的应用范围和效果。本文通过对水稻原生质体瞬时转化、甲醛固定、免疫沉淀核酸的超声波破碎等条件的优化,建立了基于水稻原生质体的染色质免疫共沉淀技术体系(chromatin immunoprecipitation system based on rice protoplasts,ChIP-RP);并通过本技术体系验证了水稻转录因子OsNF-YA4蛋白对靶基因序列的富集作用。本技术体系将减少制备特异性抗体或构建稳定遗传材料的局限,有利于水稻转录因子直接调控靶基因的快速筛选和验证,推动水稻转录因子调控功能的分子机制解析。
