胱硫醚-β-合成酶OsCBSX4调控水稻稻瘟病抗性的机理  

The Mechanism of Cystathionine-β-Synthase OsCBSX4 in Rice Blast Resistance

作　　者：刁志娟[1] 陈立喆 王勋 鲁玲 刘燕 张静[1] 夏娜 唐定中 李生平[1,2] DIAO ZhiJuan;CHEN LiZhe;WANG Xun;LU Ling;LIU Yan;ZHANG Jing;XIA Na;TANG DingZhong;LI ShengPing(College of Life Science,Fujian Agriculture and Forestry University/Plant Immunity Center,Fuzhou 350002;School of Future Technology,Fujian Agriculture and Forestry University/Fujian Provincial Key Laboratory of Crop Breeding by Design,Fuzhou 350002)

机构地区：[1]福建农林大学生命科学学院/植物免疫研究中心,福州350002 [2]福建农林大学未来技术学院/福建省作物设计育种重点实验室,福州350002

出　　处：《中国农业科学》2024年第23期4593-4606,I0001,共15页Scientia Agricultura Sinica

基　　金：科技创新2030重大项目(2023ZD04070)。

摘　　要：【目的】稻瘟病是影响水稻产量和品质的主要病害之一,OsBSK1-2在水稻稻瘟病抗性中发挥着重要作用。前期通过对OsBSK1-2进行互作蛋白筛选,获得候选蛋白胱硫醚-β-合成酶OsCBSX4。验证OsBSK1-2与OsCBSX4之间的相互作用,解析OsCBSX4在水稻抵抗稻瘟病中的功能及作用机制,为水稻抗病育种提供重要的理论基础。【方法】利用免疫共沉淀、双分子荧光互补和荧光素酶互补试验验证OsBSK1-2与OsCBSX4之间的相互作用;利用实时荧光定量PCR和农杆菌介导的烟草瞬时转化,分析OsCBSX4的表达特征及蛋白定位;利用CRISPR/Cas9和农杆菌介导的遗传转化技术构建OsCBSX4敲除和过量表达植株,并通过接种稻瘟菌进行稻瘟病抗性鉴定;同时,通过ROS爆发测定和DAB染色分析,明确oscbsx4突变体中稻瘟菌和chitin诱导的免疫反应变化;此外,利用双分子荧光互补和荧光素酶互补试验验证OsCBSX4与OsRbohB之间的相互作用,用离体接菌法分析OsCBSX4的代谢产物对水稻抵抗稻瘟菌的影响,揭示OsCBSX4的免疫功能和作用机制。【结果】免疫共沉淀、荧光素酶互补及双分子荧光互补试验均表明OsBSK1-2与OsCBSX4之间存在相互作用。敲除OsCBSX4降低了水稻对稻瘟菌的抗性,喷雾接种稻瘟菌后,与野生型相比,oscbsx4突变体病斑数量更多;打孔接种稻瘟菌后,oscbsx4突变体较野生型病斑面积更大,稻瘟菌生长量更多。同时,OsCBSX4的功能缺失也降低了稻瘟菌侵染所诱导的病程相关基因OsRP5、OsPR10的表达和H2O2的积累,以及chitin诱导的ROS爆发。而过量表达OsCBSX4则提高了水稻的稻瘟病抗性,打孔接菌后,与野生型相比,过量表达OsCBSX4植株病斑面积更小,稻瘟菌生长更少。同时,OsCBSX4能够与介导水稻ROS产生的关键蛋白OsRbohB互作。此外,用OsCBSX4的代谢产物L-胱硫醚处理水稻,并不影响水稻的稻瘟病抗性。【结论】OsCBSX4为OsBSK1-2信号通路中的重要组分,正�【Objective】Rice blast is one of the main diseases that threaten rice yield and quality.OsBSK1-2 has been found to be involved in rice blast resistance regulation.Previously,OsCBSX4,a cysteine sulfide-β-Synthase,was identified by screening the proteins that interact with OsBSK1-2.The purpose of this study was to verify the interaction between OsBSK1-2 and OsCBSX4,and clarify the function and molecular mechanism of OsCBSX4 in rice blast resistance,providing a theoretical basis for rice disease resistance breeding.【Method】Co-immunoprecipitation,bimolecular fluorescence complementation and luciferase complementation assays were used to determine the interaction between OsBSK1-2 and OsCBSX4.Then,quantitative PCR and agrobacteriummediated transient transformation in N.benthamiana were used to detect the gene expression pattern and protein localization of OsCBSX4.Subsequently,OsCBSX4-knockingout and OsCBSX4-overexpressing plants were generated via CRISPR/Cas9technology and Agrobacterium-mediated genetic transformation,respectively,and their resistance to rice blast was determined by inoculation with M.oryzae.Moreover,the immune responses induced by chitin and M.oryzae in the oscbsx4 mutant were analyzed using ROS burst and DAB staining assays respectively.In the end,the interaction between OsCBSX4 and OsRbohB was verified via bimolecular fluorescence complementation and split-luciferase complementation imaging assays,and the impact of the metabolite of OsCBSX4 on rice blast resistance was determined using the detached leaf inoculation method.The studies above will reveal the immune function and molecular mechanism of OsCBSX4.【Result】The interaction between OsCBSX4 and OsBSK1-2 was verified by CoIP,LCI and BiFC assays.Compared to the wild type,the oscbsx4 mutant showed more disease lesions after inoculation with M.oryzae by the spray method,as well as a larger lesion area and greater fungal growth after inoculation with M.oryzae by the punch method,suggesting that knocking out OsCBSX4 decreased rice blast re

关 键 词：水稻 胱硫醚-Β-合成酶 OsCBSX4 OsBSK1-2 OsRbohB 稻瘟病 免疫共沉淀 双分子荧光互补 

分 类 号：S435.111.41[农业科学—农业昆虫与害虫防治]