生物谷報道：2007年8月12日，我所柴繼杰實(shí)驗(yàn)室在Nature雜志上在線發(fā)表題為 “The structural basis for activation of plant immunity by bacterial effector protein AvrPto” 的文章,。該文章報道了第一個細(xì)菌效應(yīng)蛋白和植物中對應(yīng)的抗性蛋白的復(fù)合物AvrPto-Pto的晶體結(jié)構(gòu),，基于該結(jié)構(gòu)和相關(guān)實(shí)驗(yàn)結(jié)果，提出了AvrPto通過解除Pto對防御響應(yīng)的抑制引發(fā)疾病抗性的機(jī)制,。
植物的抗性蛋白精確識別病原菌中的效應(yīng)蛋白,對引發(fā)植物防御響應(yīng)非常重要,。利用蛋白質(zhì)晶體學(xué)、生物化學(xué)和遺傳學(xué)的方法,，研究了番茄中的抗性蛋白-蛋白激酶Pto與丁香假單胞桿菌中的效應(yīng)蛋白AvrPto的復(fù)合物,，從分子水平上揭示了細(xì)菌效應(yīng)蛋白AvrPto激活植物免疫系統(tǒng)的結(jié)構(gòu)基礎(chǔ)。該研究對深入探討植物如何識別病原菌的效應(yīng)蛋白啟動防御反應(yīng),、限制病原菌繁殖的復(fù)雜抗病機(jī)制有重要的意義,。
運(yùn)用克隆、表達(dá)和純化技術(shù)制備了AvrPto-Pto復(fù)合物,，并首次生長出合適的晶體,，然后用MAD方法測定了其晶體結(jié)構(gòu)。該復(fù)合物的晶體結(jié)構(gòu)揭示了AvrPto 與Pto 相互作用通過兩個界面調(diào)節(jié),。遺傳學(xué)與生物化學(xué)實(shí)驗(yàn)表明AvrPto通過解除Pto 對防御的抑制作用而引起抗病反應(yīng),。生化實(shí)驗(yàn)研究結(jié)果顯示AvrPto的活性loop 的磷酸化對Pto識別AvrPto 具有非常重要的調(diào)節(jié)作用。結(jié)構(gòu)比對表明AvrPto 作為Pto 的假底物與Pto 結(jié)合,，AvrPto可能是Pto 激酶的抑制劑,。生化實(shí)驗(yàn)進(jìn)一步證明：在體外，AvrPto 的確可以抑制Pto 的激酶活性,，提示AvrPto 在易感的植物內(nèi)通過抑制蛋白激酶的活性發(fā)揮其毒性功能,，而Pto 可能進(jìn)化來模擬AvrPto 的毒性目標(biāo)從而阻斷其毒性功能。
博士生邢維滿為本論文的第一作者,，論文的其他作者還有鄒艷,、劉佳凝、陳涉,、羅熙,、周儉民博士，康奈爾大學(xué)的劉群博士,、黃清秋,、郝權(quán)博士，生物物理所的畢汝昌研究員、遺傳所的朱立煌研究員,、清華大學(xué)的吳嘉煒博士,。柴繼杰博士為本文通訊作者。此項(xiàng)研究為科技部863和北京市科委資助課題,，在北京生命科學(xué)研究所完成,。（北京生命科學(xué)研究所）
原始出處:
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: [email protected]).
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.
