生物谷报道：2007年8月12日，我所柴继杰实验室在Nature杂志上在线发表题为 “The structural basis for activation of plant immunity by bacterial effector protein AvrPto” 的文章。该文章报道了第一个细菌效应蛋白和植物中对应的抗性蛋白的复合物AvrPto-Pto的晶体结构，基于该结构和相关实验结果，提出了AvrPto通过解除Pto对防御响应的抑制引发疾病抗性的机制。
植物的抗性蛋白精确识别病原菌中的效应蛋白,对引发植物防御响应非常重要。利用蛋白质晶体学、生物化学和遗传学的方法，研究了番茄中的抗性蛋白-蛋白激酶Pto与丁香假单胞杆菌中的效应蛋白AvrPto的复合物，从分子水平上揭示了细菌效应蛋白AvrPto激活植物免疫系统的结构基础。该研究对深入探讨植物如何识别病原菌的效应蛋白启动防御反应、限制病原菌繁殖的复杂抗病机制有重要的意义。
运用克隆、表达和纯化技术制备了AvrPto-Pto复合物，并首次生长出合适的晶体，然后用MAD方法测定了其晶体结构。该复合物的晶体结构揭示了AvrPto 与Pto 相互作用通过两个界面调节。遗传学与生物化学实验表明AvrPto通过解除Pto 对防御的抑制作用而引起抗病反应。生化实验研究结果显示AvrPto的活性loop 的磷酸化对Pto识别AvrPto 具有非常重要的调节作用。结构比对表明AvrPto 作为Pto 的假底物与Pto 结合，AvrPto可能是Pto 激酶的抑制剂。生化实验进一步证明：在体外，AvrPto 的确可以抑制Pto 的激酶活性，提示AvrPto 在易感的植物内通过抑制蛋白激酶的活性发挥其毒性功能，而Pto 可能进化来模拟AvrPto 的毒性目标从而阻断其毒性功能。
博士生邢维满为本论文的第一作者，论文的其他作者还有邹艳、刘佳凝、陈涉、罗熙、周俭民博士，康奈尔大学的刘群博士、黄清秋、郝权博士，生物物理所的毕汝昌研究员、遗传所的朱立煌研究员、清华大学的吴嘉炜博士。柴继杰博士为本文通讯作者。此项研究为科技部863和北京市科委资助课题，在北京生命科学研究所完成。（北京生命科学研究所）
原始出处:
Nature advance online publication 12 August 2007 | doi:10.1038/nature06109; Received 5 July 2007; Accepted 24 July 2007; Published online 12 August 2007
The structural basis for activation of plant immunity by bacterial effector protein AvrPto
Weiman Xing1,2, Yan Zou1,3,6, Qun Liu4,6, Jianing Liu1, Xi Luo1, Qingqiu Huang3, She Chen1, Lihuang Zhu3, Ruchang Bi2, Quan Hao4, Jia-Wei Wu5, Jian-Min Zhou1 & Jijie Chai1
National Institute of Biological Sciences, No. 7 Science Park Road, Beijing 102206, China
Institute of Biophysics,
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
Cornell High-Energy Synchrotron Source, Cornell University, Ithaca, New York 14853, USA
Department of Biological Sciences and Biotechnology, Tsinghua University, 100084, Beijing, China
These authors contributed equally to this work.
Correspondence to: Jijie Chai1 Correspondence and requests for materials should be addressed to J.C. (Email: chaijijie@nibs.ac.cn).
Pathogenic microbes use effectors to enhance susceptibility in host plants. However, plants have evolved a sophisticated immune system to detect these effectors using cognate disease resistance proteins1, a recognition that is highly specific, often elicits rapid and localized cell death, known as a hypersensitive response, and thus potentially limits pathogen growth2, 3, 4, 5. Despite numerous genetic and biochemical studies on the interactions between pathogen effector proteins and plant resistance proteins, the structural bases for such interactions remain elusive. The direct interaction between the tomato protein kinase Pto and the Pseudomonas syringae effector protein AvrPto is known to trigger disease resistance and programmed cell death6, 7 through the nucleotide-binding site/leucine-rich repeat (NBS-LRR) class of disease resistance protein Prf8. Here we present the crystal structure of an AvrPto–Pto complex. Contrary to the widely held hypothesis that AvrPto activates Pto kinase activity, our structural and biochemical analyses demonstrated that AvrPto is an inhibitor of Pto kinase in vitro. The AvrPto–Pto interaction is mediated by the phosphorylation-stabilized P+1 loop and a second loop in Pto, both of which negatively regulate the Prf-mediated defences in the absence of AvrPto in tomato plants. Together, our results show that AvrPto derepresses host defences by interacting with the two defence-inhibition loops of Pto.
