植物天然免疫性研究进展及其对作物抗病育种的可能影响

ISSN 0496-3490; CODEN TSHPA9

http://www.chinacrops.org/zwxb/ E-mail: xbzw@chinajournal.net.cn DOI: 10.3724/SP.J.1006.2011.00935

植物天然免疫性研究进展及其对作物抗病育种的可能影响

赵开军1,2,* 李岩强1,3 王春连1,2 高 英1,2

中国农业科学院作物科学研究所 / 农业部作物遗传育种重点实验室, 北京100081; 2农作物基因资源与基因改良国家重大科学工程, 北京100081; 3中国农业科学院研究生院, 北京100081

1

摘 要: 植物定植在充满各种病原菌的环境中却能健康生长, 显示其拥有一套免疫系统以应对病原物的侵染。最近, 人们发现植物免疫系统至少包括2个层次: 第一层为病原相关分子模式(PAMP)激发的免疫性(PTI), 即植物通过细胞表面模式识别受体(PRRs)对病原菌的PAMPs进行分子识别, 从而启动植物的防卫反应; 第二层为病原菌效应子激发的免疫性(ETI), 即有些毒性强的病原菌通过产生效应子(effectors)来抑制PTI, 从而突破植物的第一道防线, 而植物又进化出新的分子受体(例如R基因编码的NBS-LRR蛋白质)以侦察病原菌效应子并启动第二道防卫反应。数亿年来, 病原菌的侵染和植物的防卫交替进行, 促进了病原菌和植物基因组的共进化。最新的研究还发现, 黄单胞杆菌TAL effectors和寄主植物DNA 的相互识别中, 利用了精准的分子密码。TAL effector类蛋白识别植物靶基因的启动子序列, 识别模式是2个氨基酸识别一个核苷酸。通过这种识别, TAL effector操控植物靶基因的表达, 引起寄主植物的感病或抗病反应。上述抗病分子机制研究的突破, 将对植物抗病育种产生重要影响。 关键词: 植物天然免疫; TAL效应子; 植物-病原菌相互作用; 分子识别密码; 抗病育种

Recent Findings in Plant Innate Immunity and Possible Impacts on Crop Dis-ease-resistance Breeding

ZHAO Kai-Jun1,2,*, LI Yan-Qiang1,3, WANG Chun-Lian1,2, and GAO Ying1,2

1

Key Laboratory of Crop Genetics and Breeding, Ministry of Agriculture / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences,

Beijing 100081, China; 2 National Key Facility for Crop Gene Resources and Genetic Improvement, Beijing 100081, China; 3 Graduate School of the Chinese Academy of Agricultural Sciences, Beijing 100081, China

Abstract: Plants have been successfully living in such an environment in which there are myriads of potential microbial patho-gens, indicating that plants possess an efficient immunity system. Recent studies have revealed that the plant immunity system consists of two layers of defense. The first layer, based on the sensitive perception of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs) at the plant cell surface, is named as PAMP-triggered immunity (PTI). The second is called effector-triggered immunity (ETI), in which plants use additional receptors (such as R-gene products) to perceive pathogen virulence effectors that have evolved to suppress PTI. The conventional gene-for-gene resistance in plants belongs actu-ally to ETI. For millions of years, natural selection has been driving pathogens to avoid ETI either by diversifying the recognized effectors or by acquiring additional effectors that suppress ETI. On the other hand, natural selection favors plant new R-genes that can recognize the newly acquired effectors in pathogen, resulting in new ETI to be triggered again. The latest studies have re-vealed the simple cipher that governs DNA recognition by TAL (transcription activator-like) effectors from plant pathogenic Xanthomonas. TAL effectors can specifically bind the target DNA of host plant with a novel protein-DNA binding pattern in which two amino acids recognize one nucleotide. Using this recognition code, TAL effectors can bind the promoter of target genes and induce the host diseases or resistance responses. Recent findings about plant innate immunity are reviewed in this paper and their possible applications in plant breeding for disease resistance are discussed.

Keywords: Plant innate immunity; TAL-effectors; Plant-pathogen interaction; Recognition code; Plant breeding for disease resistance

大约在35亿年前, 地球上进化出细菌, 约15亿年前出现真菌, 而高等植物只有4亿多年的历史[1-2]。

因此, 植物从诞生以来, 就一直生活在充满多种潜在病原微生物的环境中, 但植物至今仍然欣欣向荣

本研究由国家转基因生物新品种培育科技重大专项(2011ZX08001-002)资助。

*

通讯作者(Corresponding author): 赵开军, E-mail: zhaokj@mail.caas.net.cn

Received(收稿日期): 2010-12-06; Accepted(接受日期): 2011-03-06.

936

作 物 学 报 第37卷

地生长, 这表明, 植物为了防御病原菌的入侵, 已进化出一套天然的免疫系统[3-4]。随着分子生物学的飞速发展, 特别是近年对多种病原菌及其寄主植物基因组的测序以及植物抗病分子机制的研究, 不断揭示植物免疫系统的分子奥秘。最近, 科学家已发现黄单胞杆菌与寄主植物互作识别的分子密码[5-6]。本文综述了近年关于植物抗病分子机制的研究进展, 并讨论其可能对作物抗病育种产生的影响。

1 植物对病原菌侵染的基础抗性(PTI)

与哺乳动物不同, 植物定植于土壤中不能移动, 植株体内既没有可移动的防卫细胞, 也缺乏体细胞适应性的免疫系统。植物依靠细胞先天免疫力以及从病菌侵染点发出的系统信号传导来抵御病原菌的侵袭[7-10]。

近年的研究发现, 每种病原菌都具有其保守的分子特征, 例如细菌的鞭毛蛋白(flagellin), 那些保守的分子特征被称为病原相关分子模式(pathogen- associated molecular patterns, PAMPs)[7]。在病原菌刚刚与植物接触的瞬间, 植物通过其细胞表面的模式识别受体(pattern recognition receptors, PRRs)来感知病原菌的PAMPs, 从而识别各类微生物, 例如通过感知几丁质来识别真菌, 通过感知鞭毛蛋白来识别细菌。近十年来, 科学家发现植物对微生物病原相关分子模式的识别(pattern recognition, PR)是植物免疫的最基本过程[11]。这种通过植物模式识别受体感知PAMPs并启动的主动防卫反应被定义为植物的基础抗性(basal disease resistance), 也称为基础免疫(basal immunity)[11]。人们还发现植物基础抗性与传统病理学的概念紧密联系, 因此建议将传统的水平抗性(horizontal disease resistance)和基础抗性统一为PAMP激发的免疫, 即PTI (PAMP-triggered immu-nity)[7,9,11]。PTI可以激活植物的一系列抗病反应, 包括胼胝质沉积、激酶的活化、PR-蛋白的表达以及小RNA的合成等[12], 从而阻止环境中绝大部分病原菌的入侵[11](图1)。目前已证明PTI在植物抗病免疫系统发挥着十分重要的作用[13-15]。

植物模式识别受体的特点是它的高度灵敏性和专化性, 植物如果拥有某种PRR, 即使PAMP的处理浓度在纳摩尔以下, 也能感知到相应微生物对应的PAMPs, 相反, 植物缺乏这种PRR, 它就不能识别该微生物[7]。对于病原菌的PAMP, 其某个功能保守的区域往往是植物PRR识别的位点。例如细菌鞭

毛蛋白N端的22个氨基酸就是植物模式识别受体FLS2的识别位点。该识别位点的突变可以使其逃脱

拟南芥FLS2的识别[16]。从基因组序列比对分析,

FLS2基因在植物中是高度保守的, 例如番茄中存在一个FLS2的同源基因LeFLS2, 但LeFLS2特异性识别大肠杆菌鞭毛蛋白的保守多肽flg15, 而不能识别丁香假单胞菌的鞭毛蛋白flg22[17]; 实验已证明水稻的FLS2同源基因亦行使识别细菌鞭毛蛋白的功能[18]。所以FLS2的同源基因在进化上是非常古老和保守的。

另一个研究得比较清楚的模式识别受体是拟南芥的EFR (EF-Tu receptor), 它特异识别细菌的延伸因子EF-Tu (elongation factor Tu)[19], 但这种识别反应仅限于十字花科植物, 在其他植物中并未发现[20]。然而, 目前已完成基因组测序的植物都含有EFR的同源基因, 编码的蛋白质具有类似的LRR (leu-cine-rich repeat)结构[11]。在水稻基因组中, 与EFR类似含有LRR结构的基因有40多个, 其中之一就是抗白叶枯病基因Xa21。最近, Lee等[21]已从白叶枯病菌中克隆到编码对应于Xa21的PAMPs基因Ax21 (Activator of XA21-mediated immunity)。Ax21为一个含194个氨基酸的蛋白, 其中N末端的17个氨基酸(Y22为硫酸化的酪氨酸)的寡肽axYs22, 是其活性所必需的。axYs22在黄单胞菌属中是100%保守的, 因此Lee等[21]认为Ax21是一类PAMPs分子, 而XA21则为模式识别受体。现在看来, 不同种类植物的EFR-类PRRs虽然在序列上有一定的保守性, 但它们很可能识别不同的PAMPs。

真菌几丁质也是一种PAMP分子, 目前已从水稻中分离出结合几丁质的模式识别受体基因CEBiP, 其编码的跨膜糖蛋白含有两个胞外LysM结构域, 但是缺乏胞内激酶域[22], 而水稻中的另一个几丁质识别受体OsCERK1却是一个跨膜受体激酶, 并与CEBiP协同进行几丁质识别信号传导[23]。拟南芥中也存在其同源基因[24]。最近, Chen等[25]详细分析了水稻几丁质识别受体OsCERK1在成熟、运输、质膜定位及识别几丁质信号等方面的蛋白网络。

病原菌另外的几个PAMPs在生化上已很清楚, 但其受体还没有鉴定出来。它们有细菌蛋白类PAMPs, 包括具有RNP-1保守结构域的冷激蛋白[26]、超氧化物歧化酶[27]、对应Xanthomonas harpin HpaG的23-氨基酸的肽链[28]; 卵菌(Oomycetes)蛋白类PAMPs, 如转谷氨酰氨酶残基pep13[29]等; 亲脂类PAMPs, 包括二十碳四烯酸[30]、麦角固醇[31]; 细菌

第6期

赵开军等: 植物天然免疫性研究进展及其对作物抗病育种的可能影响 937

图1 植物免疫系统

Fig. 1 A model of plant immunity system

植物防御系统可大致分为两层。第一层为病原相关分子模式激发的免疫(PTI), 也称基础抗性。植物首先利用细胞质膜上的模式识别受体识别病原微生物的病原相关分子模式, 从而引起抗病反应。第二层防御系统称为效应子激发的免疫反应(ETI)。一些致病力强的病原菌通过向寄主分泌毒性因子如冠菌素、胞外多糖及蛋白类效应子等以抑制植物的基础抗性, 植物则利用NBS-LRR类R蛋白直接或间接地识别效应子, 并引起一系列防卫反应, 病原菌的生长与扩展。病原菌在自然选择的压力下进化出新的效应子, 以避开植物R蛋白的识别。植物也相应地进化出新的R基因以识别新的效应子, 于是ETI再一次被激发, 这样便促进了病原菌与寄主植物的共进化。

The plant immunity system consists of roughly two layers of defense. The first layer is named PTI (PAMP-triggered immunity), also called basal disease resistance. The plants can perceive pathogen- associated molecular patterns (PAMPs ) through pattern recognition receptors (PRRs) at the plant’s cell surface and trigger the subsequent immunity response. The second layer of defense is called effector-triggered im-munity (ETI). Successful pathogens deliver virulence factors, including the phytotoxin coronatine, extracellular polysaccharides, and pro-teinaceous effectors into the host to inhibit the PTI. The second layer of the immunity is based on the direct or indirect perception of pathogen effecors through the typically nucleotide binding leucine-rich repeat (NBS-LRR) R proteins, resulting in a series of defense response to re-strict the pathogen growth and dispersal. The pathogen can evolve new effectors to infect plants under the pressure of natural selection, which also favours new plant NBS-LRR alleles that can recognize one of the newly acquired effectors, resulting again in ETI. All of this promote

the coevolution of the pathogen microbes and host plants.

细胞壁肽聚糖[32]; 革兰氏阴性菌脂寡糖(lipooli- 胞菌的另外一个效应蛋白HopAI1也有抑制PTI的gosaccharide)[33]。另外还有些待确认的PAMPs, 包括细菌的群体密度感应信号quorum sensing signal[34]和真菌的Fe载体siderophores[35]等。

功能, HopAI1是一个磷酸苏氨酸(phosphothreonine)裂解酶, 使丝裂原活化蛋白激酶(mitogen-activated protein kinases, MAPKs)MPK3和MPK6去磷酸化, 从而终止PRR的信号传导[43]。

病原菌除了通过其效应子直接与植物的PRRs或MAPKs互作来抑制植物的PTI, 还利用效应子干扰PRR信号传导的下游环节或其他抗病相关的通路。例如, 丁香假单胞菌的效应子HopU1通过腺苷二磷酸核糖基化来修饰拟南芥的RNA 结合蛋白GRP7等; 其另一个效应子HopM1可以诱导拟南芥MIN7蛋白的降解, 而MIN7蛋白对植物的抗性非常重要[44]。最近, Wang等[45]发现丁香假单胞菌的效应子HopF2在拟南芥中可以与寄主MKK5互作而阻断寄主的PTI防卫反应。总之, 近年功能基因组研究揭示, 细菌、真菌、卵菌及线虫分泌到寄主植物细胞中的效应子估计达百种以上[4]。

2 植物对病原菌的基因对基因抗性(ETI)

所有的病原菌都带有PAMPs, 而每种植物都会遭受某些病原菌的感染, 说明一些病原菌能够克服植物的PTI。目前已知, 为了自身的定植与生长, 各种病原菌, 包括细菌[36]、真菌[37]和卵菌[38], 都向其寄主植物细胞注入毒性因子(virulence factors)来抑制植物的基础抗性即PTI (图1)。例如病原细菌通过III型分泌系统将蛋白类效应子等注入植物细胞以抑制PTI [7]。丁香假单胞菌株DC3000分泌的效应蛋白AvrPto和AvrPtoB 与FLS2和BAK1的激酶域进行直接互作从而抑制激酶活性[39]或阻止FLS2-BAK1丁香假单复合体的形成[40-42], 从而抑制植物的PTI。

938

作 物 学 报 第37卷

病原菌也合成一些低分子毒素来攻击植物的PTI防线, 例如丁香假单胞菌株 DC3000合成冠菌素(coronatin)模拟JA以干扰植物的PTI防卫[46], 合成丁香花素(syringolin)抑制植物的蛋白酶[47]。一些病原菌在寄生于植物细胞间隔时会产生胞外多糖EPS (extracellular polysaccharides), 与钙离子螯合, 从而影响PAMPs-PRRs识别过程中的钙信号传导[48]。

病原菌通过产生效应子等来克服植物的基础抗性或第一道防线, 植物又进化出基于R-基因的第二道防线: 植物通过新的分子受体(例如R基因编码的NBS-LRR蛋白质)来发现这些效应子并防卫。这种通过R-基因蛋白识别病原菌效应子并启动的主动防卫反应被称为植物的R-基因抗性(R-gene-based dis-ease resistance)[7]。Jones和Boller等建议将传统的植物垂直抗性(horizontal disease resistance)和R-基因抗性统一为效应子激发的免疫, 即ETI (effector triggered immunity)[4,7,10-11]。PTI是植物对一类含有PAMPs的病原菌的识别, 范围更宽泛, 故作为植物基础免疫; 与PTI不同, ETI为一类效应子激发的由抗病基因介导的植物免疫, 是含有抗病基因的植物识别含有相应效应子的病原菌引起的免疫反应。

病原菌的上述效应子是菌种甚至小种特有的。植物识别病原菌效应子的受体绝大多数是NBS-LRR蛋白, 在拟南芥中有近150个基因编码这类蛋白[49]。Caplan等最近对这类蛋白的结构及其在信号识别中的功能进行了综述[50]。有些NBS－LRR蛋白质直接与效应子蛋白互作[37], 但更多是作为警卫(guard)与效应子蛋白间接互作[51]。植物通过识别病原菌效应子蛋白, 激发一系列的抗病防卫反应, 如果防卫反应非常强烈, 会在侵染点引起寄主细胞程序性死亡, 即过敏性反应(hypersensitive reaction, HR), 从而病原菌的生长、繁殖和扩张[50]。病原菌在自然选择的压力下进化出新的效应子, 以克服植物初级的ETI, 而植物也相应地进化出新的R基因以应对病原菌进化的新的效应子, 于是新的ETI再一次被激发, 这样便促进了病原菌与寄主植物基因组的共进化[7], 因此植物的ETI防线包含多个亚层次(图1)。

3 植物和病原菌相互识别的分子密码

总体上讲, 不论是植物细胞表面模式识别受体(PRRs)对PAMPs的识别, 还是植物R基因编码蛋白与病原菌效应子(effectors)的识别, 人们对其分子互作的细节还知之甚少。但近年关于黄单胞杆菌的蛋

白类效应子(Transcription activator-like effector, TAL effector)与寄主植物基因互作研究取得了许多新进展[52-57], 其中最令人激动的是病原菌TAL effector特异性识别植物靶基因的分子密码得以破解[5-6]。

黄单胞杆菌属(Xanthomonas)的多个致病变种, 在多种作物上造成严重的病害, 例如辣椒和番茄的细菌性斑点病(Xanthomonas campestris pv. vesicato-ria)、水稻的白叶枯病(Xanthomonas oryzae pv. oryzae)和条斑病(Xanthomonas oryzae pv. oryzicola)。黄单胞杆菌通过III型分泌系统将其效应子蛋白TAL ef-fector注入寄主细胞中, 并模拟真核转录因子操纵寄主细胞的基因表达[54-57]。TAL effectors介导的一些基因表达, 能够引起植物生理状态上的变化, 例如细胞肿胀[54], 从而使寄主体内环境更有利于病原菌的增殖和扩散。水稻白叶枯病原菌的TAL effectors PthXo1和PthXo6分别作用于水稻的感病基因Os8N3和感病相关的转录因子基因OsTFX1, 引起水稻感病[56-57]。而植物亦进化出能够识别相应TAL effectors的抗病基因, 例如水稻的Xa27抗病基因识别白叶枯病菌TAL effector AvrXa27并引起一系列的抗病反应[58]。我们也发现白叶枯病菌中无毒蛋白AvrXa23 (一个TAL effector)被水稻Xa23基因的启动子陷阱捕获而产生强烈的抗病反应(中国发明专利申请号201010256332.8)。

从氨基酸序列看, TAL effectors包括由多个串联重复单元组成的串联重复区中心结构域、C端的核 定位信号(NLSs)和酸性激活结构域(AD)(图2-A)[59-60]。不同TAL effectors的氨基酸序列高度保守, 其主要差异在串联重复区, 重复单元的数目和顺序决定了TAL effectors的特异性[61-62], 及对寄主植物品种的专化性。

AvrBs3是TAL effector家族中的典型成员, 最先发现于Xanthomonas campestris pv. vesicatoria (Xcv)[63]。AvrBs3直接识别寄主靶基因启动子的UPA (upregulated by AvrBs3) box元件, 引起UPA基因的表达, 其中包括辣椒中抗病基因Bs3的表达[54-55]。AvrBs3的串联重复区由17.5个几乎完全相同的重复单元组成; 每个重复单元含有34个氨基酸, 其中的第12和第13个氨基酸是高度可变的(图2-A)[5]。最近Bonas等[5]依据这些重复可变的第12和第13个氨基酸的不同进行了归类, 发现AvrBs3的每一个重复单元识别寄主靶基因启动子UPA box中一个特异的DNA碱基, 且这种识别由每一个重复单元的第

第6期

赵开军等: 植物天然免疫性研究进展及其对作物抗病育种的可能影响 939

图2 TAL effectors特异性结合靶基因DNA的分子模型 Fig. 2 Model for DNA target specificity of TAL effectors

[5] [6]

根据Boch等(2009)和Moscou等(2009)设计。A: TAL effectors包含中心串联重复区, 核定位信号(NLS), 以及酸性激活结构域(AD)。图中显示了AvrBs3的第一个重复单元的氨基酸序列。其中第12和第13位氨基酸为高度可变区[5]。B: 将AvrBs3中17.5个重复单元的第12和第13位氨基酸与靶基因相对应的UPA box进行比对[5]。C: 根据如下已有的效应子中重复可变区氨基酸与靶基因DNA的组合: AvrXa27/Xa27、AvrBs3/Bs3、AvrBs3/UPA20、AvrBs3∆rep16/Bs3E、AvrBs3∆rep109/Bs3、AvrHah1/Bs3、PthXo6/OsTFX1、PthXo7/Os TFIIAγ1和Tal1c/OsHEN, 以及10种利用40个新增的TAL effectors 的重复区与受TAL effectors诱导表达的水稻基因启动子的目标序列进行人工扫描比对的结果, 得到了DNA的正义链与相对应重复的可变氨基酸的对应关系。* 表示该重复的13位氨基酸空缺。粗字体表示最高频数的氨基酸[6]。

Modified from Boch et al. [5] (2009) and Moscou et al. [6] (2009). A: TAL effectors contain central tandem repeats (repeat domain), nuclear localization signals (NLSs), and an acidic transcriptional activation domain (AD). Shown is the amino acid sequence of the first repeat of AvrBs3. The amino acids 12 and 13 are hypervariable [5]. B: Hypervariable amino acids at position 12 and 13 of the 17.5 AvrBs3 repeats are aligned to the UPA box consensus [5]. C: Nucleotides in the upper DNA strand that correspond to the hypervariable amino acids in each repeat

are counted on the basis of the following combinations of nine effectors and experimentally identified target genes: AvrXa27/Xa27, AvrBs3/Bs3, AvrBs3 /UPA20, AvrBs3∆rep16/Bs3E, AvrBs3∆rep109/Bs3, AvrHah1 /Bs3, PthXo6/OsTFX1, PthXo7/OsTFIIAγ1 and

Tal1c/OsHEN. And 10 more alignments obtained by scanning all rice promoters with 40 additional X. oryzae TAL effectors, retaining for each effector the best alignment for which the downstream gene is activated during infection. An asterisk indicates that amino acid 13 is missing in

this repeat type. The highest nucleotide frequencies are in bold [6].

12和第13位氨基酸残基决定, 例如HD和NI分别对C和A有很强的偏爱(图2-B)。通过分析多个TAL effectors的重复类型与其靶基因启动子区UPA box的对应关系, Boch和Bonas等[5]构建了TAL effectors不同重复类型特异性识别目标DNA碱基的分子密码模型(图2-C)。同时, Moscou和Bogdanove[6]利用计算机扫描比对TAL effectors中可变重复类型与目标启动子中的DNA序列, 也得到了类似的分子密码模型。目前, 已有更新的研究验证了这种分子密码的可靠性[-65]。

疫系统完全可以应对病原菌的侵染和变异, 而且可以想象, 在完全自然的条件下, 植物与病原微生物之间会达成一种相互共存的平衡。但是从农业生产的层面看, 农作物新品种抵御病原菌侵染的能力非常脆弱, 常常表现出大幅度的减产而被淘汰。所以长期以来, 作物抗病育种备受重视, 而且效果显著。但是随着作物单产的不断提高和抗性资源的消耗, 作物抗病性的提高已变得越来越困难。那么近年植物抗病分子机理研究的进展, 将对作物抗病育种产生哪些影响？现予以讨论。

必须重视PTI的利用。既然已发现植物对微生物病原相关分子模式的识别是植物免疫的最基本过程, 并证明PTI在植物抗病免疫系统发挥着十分重要的作用, 那么育种家可以考虑如何将PTI有效应

4 抗病分子机理研究进展对作物抗病育种的可能影响

从自然种群生存的角度看, 植物拥有的一套免

940

作 物 学 报 第37卷

用于作物抗病品种选育。从PTI的性质看, 它类似于传统概念的水平抗性, 但不一定受微效多基因控制。因此在利用PTI的途径上, 有可能完全按照主基因的方式操作, 条件是利用分子检测技术。最近, Lacombe等[66]利用农杆菌介导法将拟南芥识别细菌延伸因子EF-Tu的受体编码基因EFR 转入烟草和番茄基因组中, 获得的转基因植物可以抗来自假单胞菌属(Pseudomonas)、农杆菌属(Agrobacterium)、黄单胞菌属(Xanthomonas)和拉尔氏菌属(Ralstonia)等不同属的病源细菌, 充分展示了利用不同植物的模式识别受体基因培育广谱、持久抗病农作物的可能性。可以预计, 随着更多PRRs基因的克隆, 完全可以利用基因工程途径来提高作物品种的PTI抗病能力。

重视将ETI与PTI结合利用。由于病原菌的效应子是菌种甚至小种特有的, 而植物的大多数R-基因蛋白是专门为识别病原菌效应子而进化的, 植物ETI的特性是强度大但容易激发病原菌产生新的效应子从而克服原有的ETI抗性。因此在利用ETI时需要结合利用PTI, 以便达到两道关口协防的效果, 这类似于排球比赛中的前排拦网与后排保护的联防, 如此可以避免那些大面积推广品种因突然丧失ETI抗性而造成重大产量损失。在重视PTI的过程中结合ETI的使用, 主要为抗病基因的利用。通过分子标记辅助选择或使用其自身启动子的抗病基因进行转基因育种, 获得抗病植株。利用多基因聚合或多基因转化将多个优良抗病基因导入PTI抗性较好的栽培品种中, 有利于扩大作物的抗病谱, 改良作物的ETI抗性。许多抗病基因是受效应子诱导表达的, 例如在白叶枯病抗病育种中, 病原菌中的TAL ef-fector能寄主植物的基因表达。

据此可以将病原菌无毒基因和植物相应抗病基因置于病菌侵入诱导表达的启动子下, 通过基因工程将其导入寄主植物, 当寄主受到病菌侵染时, 无毒基因便被诱导表达, 无毒蛋白作为激发子, 激发其特异抗病基因表达产生对应的受体蛋白, 它们之间相互识别, 导致寄主的过敏性反应, 这样可以提高作物品种的ETI抗病能力。当然, 植物的ETI抗性和PTI抗性本身就是植物免疫系统不可分割的重要部分。只是随着植物分子病理学研究的深入才将其进行了特别的区分或细化。在作物抗病改良育种中, 随着近年来许多抗病基因的克隆, 在利用PTI基础上, 利用ETI中抗病基因的抗性, 在未来的分子育种中将有更广阔的前景。

设计广谱抗病基因复合体。TAL effector与寄主靶基因识别分子密码的破解, 揭示了一种新型的蛋白质-DNA结合方式, 这种控制蛋白质和DNA相互识别的机制可能开辟作物抗病育种新途径—构建广谱抗病基因复合体。Römer等[67]利用基因工程的方法将不同的UPA Box相互组合, 得到了能与多个TAL effectors识别的新启动子, 用这种启动子驱动一个抗病基因, 就可以扩大该抗病基因的抗谱。另外, 目前从细菌性条斑病细菌中已克隆到一些TAL effector编码基因[68], 但是抗细菌性条斑病的主效基因很少, 目前比较有效的为非寄主抗性基因Rxo1[69], 而组成型表达的抗白叶枯病基因Xa27也抗一些细菌性条斑病小种[70]。因此我们可以利用TAL effector与寄主靶基因DNA识别的分子密码设计受细菌性条斑病菌TAL effector识别的启动子, 用其驱动抗病基因Rxo1或Xa27, 以获得抗细菌性条斑病的抗病基因复合体。

References

[1] Bill B. A Short History of Nearly Everything. New York: Broad-way Books, 2003. pp 188–202

[2] Butterfield N J. Probable proterozoic fungi. Paleobiology, 2005,

31: 165–182

[3] Takken F L W, Tameling W I L. To nibble at plant resistance pro-teins. Science, 2009, 324: 744–745

[4] Boller T, He S Y. Innate immunity in plants: an arms race be-tween pattern recognition receptors in plants and effectors in mi-crobial pathogens. Science, 2009, 324: 742–744

[5] Boch J, Scholze H, Schornack S, Landgraf A, Hahn S, Kay S,

Lahaye T, Nickstadt A, Bonas U. Breaking the code of DNA binding specificity of TAL-type III effectors. Science, 2009, 326: 1509–1512

[6] Moscou M J, Bogdanove A J. A simple cipher governs DNA

recognition by TAL effectors. Science, 2009, 326: 1501

[7] Jones J D G, Dangl J L. The plant immune system. Nature, 2006,

444: 323–329

[8] Dangl J L, Jones J D G. Plant pathogens and integrated defense

responses to infection. Nature, 2001, 411: 826–833

[9] Ausubel F M. Are innate immune signaling pathways in plants

and animals conserved? Nat Immunol, 2005, 6: 973–979 [10] Chisholm S T, Coaker G, Day B, Staskawicz B J. Host-microbe

interactions: shaping the evolution of the plant immune response. Cell, 2006, 124: 803–814

[11] Boller T, Felix G. A renaissance of elicitors: perception of mi-crobe-associated molecular patterns and danger signals by pat-tern-recognition receptors. Annu Rev Plant Biol, 2009, 60: 379– 406

[12] Navarro L, Jay F, Nomura K, He S Y, Voinnet O. Suppression of

the microRNA pathway by bacterial effector proteins. Science,

第6期

赵开军等: 植物天然免疫性研究进展及其对作物抗病育种的可能影响 941

2008, 321: 9–967

[13] Göhre V, Spallek T, Häweker H, Mersmann S, Mentzel T, Boller

T, Torres M, Mansfield J W, Robatzek S. Plant pattern-recogni- tion receptor FLS2 is directed for degradation by the bacterial ubiquitin ligase AvrPtoB. Curr Biol, 2008, 23: 1824–1832 [14] Zipfel C, Robatzek S, Navarro L, Oakeley E J, Jones J D, Felix G,

Boller T. Bacterial disease resistance in Arabidopsis through flagellin perception. Nature, 2004, 428: 7–767

[15] Gómez-Gómez L, Boller T. FLS2: an LRR receptor-like kinase

involved in the perception of the bacterial elicitor flagellin in Arabidopsis. Mol Cell, 2000, 5: 1003–1011

[16] Sun W, Dunning F M, Pfund C, Weingarten R, Bent A F.

Within-species flagellin polymorphism in Xanthomonas campes-tris pv. campestris and its impact on elicitation of Arabidopsis FLAGELLIN SENSING2-dependent defenses. Plant Cell, 2006, 18: 7–779

[17] Robatzek S, Bittel P, Chinchilla D, Köchner P, Felix G, Shiu S H,

Boller T. Molecular identification and characterization of the to-mato flagellin receptor LeFLS2, an orthologue of Arabidopsis FLS2 exhibiting characteristically different perception specifici-ties. Plant Mol Biol, 2007, : 539–547

[18] Takai R, Isogai A, Seiji S, Che F S. Analysis of flagellin percep-tion mediated by flg22 receptor OsFLS2 in rice. Mol Plant- Microbe Interact, 2008, 12: 1635–12

[19] Kunze G, Zipfel C, Robatzek S, Niehaus K, Boller T, Felix G.

The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants. Plant Cell, 2004, 16: 3496–3507 [20] Zipfel C, Kunze G, Chinchilla D, Caniard A, Jones J D G. Per-ception of the bacterial PAMP EF-Tu by the receptor EFR re-stricts Agrobacterium-mediated transformation. Cell, 2006, 125: 749–760

[21] Lee S W, Han S W, Sririyanum M, Park C J, Seo Y S, Ronald P

C. A type I−secreted, sulfated peptide triggers XA21-mediated innate immunity. Science, 2009, 326: 850–853

[22] Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto-Tomiyama C,

Dohmae N. Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor. Proc Natl Acad Sci USA, 2006, 103: 11086–11091

[23] Shimizu T, Nakano T, Akamizawa D, Desaki Y, Ishii-Minami N,

Nishizawa Y, Minami E, Okada K, Yamane H, Kaku H, Shibuya N. Two LysM receptor molecules, EBiP and OsCERK1, coop-eratively regulate chitin elicitor signaling in rice. Plant J, 2010, : 204–214

[24] Miya A, Albert P, Shinya T, Desaki Y, Ichimura K. CERK1, a

LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis. Proc Natl Acad Sci USA, 2007, 104: 19613–19618 [25] Chen L, Hamada S, Fujiwara M, Zhu T, Thao N P, Wong H L,

Krishna P, Ueda T, Kaku H, Shibuya N, Kawasaki T, Shimamoto K. The Hop/Sti1-Hsp90 chaperone complex facilitates the matu-ration and transport of a PAMP receptor in rice innate immunity. Cell Host & Microbe, 2010, 7: 185–196

[26] Felix G, Boller T. Molecular sensing of bacteria in plants—the

highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco. J Biol Chem, 2003, 278: 6201–6218

[27] Watt S A, Tellstrom V, Patschkowski T, Niehaus K. Identifica-tion of the bacterial superoxide dismutase (SodM) as plant- inducible elicitor of an oxidative burst reaction in tobacco cell suspension cultures. J Biotechnol, 2006, 126: 78–86

[28] Kim J G, Jeon E, Oh J, Moon J S, Hwang I. Mutational analysis

of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants. J Bacte-riol, 2004, 186: 6239–6247

[29] Brunner F, Rosahl S, Lee J, Rudd J J, Geiler C, Kauppinen S.

Pep-13, a plant defense-inducing pathogenassociated pattern from Phytophthora transglutaminases. EMBO J, 2002, 21: 6681–6688 [30] Boller T. Chemoperception of microbial signals in plant cells.

Annu Rev Plant Physiol Plant Mol Biol, 1995, 46: 1–214 [31] Granado J, Felix G, Boller T. Perception of fungal sterols in

plants (subnanomolar concentrations of ergosterol elicit extracel-lular alkalinization in tomato cells. Plant Physiol, 1995, 107: 485–490

[32] Erbs G, Silipo A, Aslam S, De Castro C, Liparoti V, Flagiello A,

Pucci P, Lanzetta R, Parrilli M, Molinaro A, Newman M A, Cooper R M. Peptidoglycan and muropeptides from pathogens Agrobacterium and Xanthomonas elicit plant innate immunity: structure and activity. Chem & Biol, 2008, 15: 438–448

[33] Silipo A, Sturiale L, Garozzo D, Erbs G, Jensen T T, Lanzetta R,

Dow J M, Parrilli M, Newman M A, Molinaro A. The acylation and phosphorylation pattern of lipid from Xanthomonas campes-tris strongly influence its ability to trigger the innate immune re-sponse in Arabidopsis. Chem Biol Chem, 2008, 9: 6–904 [34] Dulla G, Lindow S E. Quorum size of Pseudomonas syringae is

small and dictated by water availability on the leaf surface. Proc Natl Acad Sci USA, 2008, 105: 3082–3087

[35] Johnson L. Iron and siderophores in fungal-host interactions.

Mycol Res, 2008, 112: 170–183

[36] Abramovitch R B, Anderson J C, Martin G B. Bacterial elicita-tion and evasion of plant innate immunity. Nat Rev Mol Cell Biol, 2006, 7: 601–611

[37] Ellis J G, Dodds P N, Lawrence G J. Flax rust resistance gene

specificity is based on direct resistance avirulence protein inter-actions. Annu Rev Phytopathol, 2007, 45: 2–306

[38] Kamoun S. Groovy times: filamentous pathogen effectors re-vealed. Curr Opin Plant Biol, 2007, 10: 358–365

[39] Xiang T, Zong N, Zou Y, Wu Y, Zhang J, Xing W, Li Y, Tang X,

Zhu L, Chai J, Zhou J M. Pseudomonas syringae effector AvrPto blocks innate immunity by targeting receptor kinases. Curr Biol, 2008, 18: 74–80

[40] Shan L, He P, Li J, Heese A, Peck S C, Nürnberger T, Martin G

B, Sheen J. Bacterial effectors target the common signaling part-ner BAK1 to disrupt multiple MAMP receptor-signaling com-plexes and impede plant immunity. Cell Host & Microbe, 2008, 4: 17–27

942

作 物 学 报 第37卷

[41] Chinchilla D, Zipfel C, Robatzek S, Kemmerling B, Nurnberger

T. A flagellin-induced complex of the receptor FLS2 and BAK1 initiates plant defence. Nature, 2007, 448: 497–500

[42] Heese A, Hann D R, Gimenez-Ibanez S, Jones A M E, He K, Li J,

Schroeder J I, Peck S C, Rathjen J P. The receptor-like kinase SERK3/BAK1 is a central regulator of innate immunity in plants. Proc Natl Acad Sci USA, 2002, 104: 12217–12222

[43] Zhang J, Shao F, Li Y, Cui H, Chen L, Li H, Zou Y, Long C, Lan

L, Chai J, Chen S, Tang X, Zhou J M. A Pseudomonas syringae effector inactivates MAPKs to suppress PAMP-induced immu-nity in plants. Cell Host & Microbe, 2007, 1: 175–185

[44] Block A, Li G Y, Fu Z Q, Alfano J R. Phytopathogen type III ef-fector weaponry and their plant targets. Curr Opin Plant Biol, 2008, 11: 396–403

[45] Wang Y, Li J, Hou S, Wang X, Li Y, Ren D, Chen S, Tang X,

Zhou J. A Pseudomonas syringae ADP-Ribosyltransferase inhi- bits Arabidopsis mitogen-activated protein kinase kinases. Plant Cell, 2010, 22: 2033–2044

[46] Melotto M, Underwood W, Koczan J, Nomura K, He S Y. Plant

stomata function in innate immunity against bacterial invasion. Cell, 2006, 126: 969–980

[47] Groll M, Schellenberg B, Bachmann A S, Archer C R, Huber R,

Powell T K, Lindow S, Kaiser M, Dudler R. A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism. Nature, 2008, 452: 755–758

[48] Aslam S N, Newman M A, Erbs G, Morrissey K L, Chinchilla D,

Boller T, Jensen T T, De Castro C, Ierano T, Molinaro A, Jack-son R W, Knight M R, Cooper R M. Bacterial polysaccharides suppress induced innate immunity by calcium chelation. Curr Biol, 2008, 18: 1078–1183

[49] Meyers B C, Kozik A, Griego A, Kuang H H, Michelmore R W.

Genome-wide analysis of NBS-LRR-encoding genes in Arabi-dopsis. Plant Cell, 2003, 15: 809–834

[50] Caplan J, Padmanabhan M, Dinesh-Kumar S P. Plant NB-LRR

immune receptors: from recognition to transcriptional repro-gramming. Cell Host & Microbe, 2008, 3: 126–135

[51] Lotze M T, Zeh H J, Rubartelli A, Sparvero L J, Amoscato A A,

Washburn N R, Devera M E, Liang X, Tör M, Billiar T. The grateful dead: damage associated molecular pattern molecules and reduction/oxidation regulate immunity. Immunologcal Rev, 2007, 220: 60–81

[52] Kay S, Bonas U. How Xanthomonas type III effectors manipulate

the host plant. Curr Opin Microbiol, 2009, 12: 37–43

[53] White F F, Yang B. Host and pathogen factors controlling the

rice-Xanthomonas oryzae interaction. Plant Physiol, 2009, 150: 1677–1686

[54] Kay S, Hahn S, Marois E, Hause G, Bonas U. A bacterial effector

acts as a plant transcription factor and induces a cell size regula-tor. Science, 2007, 318: 8–651

[55] Römer P, Hahn S, Jordan T, Strauss T, Bonas U, Lahaye T. Plant

pathogen recognition mediated by promoter activation of the pepper Bs3 resistance gene. Science, 2007, 318: 5–8 [56] Sugio A, Yang B, Zhu T, White F F. Two type III effector genes

of Xanthomonas oryzae pv. oryzae control the induction of the

host genes OsTFIIAγ1 and OsTFX1 during bacterial blight of rice. Proc Natl Acad Sci USA, 2007, 104: 10720–10725

[57] Yang B, Sugio A, White F F. Os8N3 is a host disease-suscep-

tibility gene for bacterial blight of rice. Proc Natl Acad Sci USA, 2006, 103: 10503–10508

[58] Gu K, Yang B, Tian D, Wu L, Wang D, Sreekala C, Yang F, Chu

Z, Wang G L, White F F, Yin Z. R gene expression induced by a type-III effector triggers disease resistance in rice. Nature, 2005, 435: 1122–1125

[59] Schornack S, Meyer A, Römer P, Jordan T, Lahaye T. Gene-

for-gene-mediated recognition of nuclear-targeted AvrBs3-like bacterial effector proteins. J Plant Physiol, 2006, 163: 256–272 [60] Van den Ackerveken G, Marois E, Bonas U. Recognition of the

bacterial avirulence protein AvrBs3 occurs inside the host plant cell. Cell, 1996, 87: 1307–1316

[61] Conrads-Strauch J H K, Bonas U. Race-specificity of plant resis-tance to bacterial spot disease determined by repetitive motifs in a bacterial avirulence protein. Nature, 1992, 356: 172–174 [62] Yang B, Sugio A, White F F. Avoidance of host recognition by

alterations in the repetitive and C-terminal regions of AvrXa7, a type III effector of Xanthomonas oryzae pv. oryzae. Mol Plant-Microbe Interact, 2005, 18: 142–149

[63] Bonas U, Stall R E, Staskawicz B. Genetic and Structural char-acterization of the avirulence gene avrBs3 from Xanthomonas campestris pv. vesicatoria. Mol Gen Genet, 19, 218: 127–136 [] Li T, Huang S, Jiang W Z, Wright D, Spalding M H, Weeks D P,

Yang B. TAL nucleases (TALNs): hybrid proteins composed of TAL effectors and FokI DNA-cleavage domain. Nucl Acids Res, 2011, 39: 359–372

[65] Christian M, Cermak T, Doyle E L, Schmidt C, Zhang F,

Hummel A, Bogdanove A J, Voytas D F. TAL effector nucleases create targeted DNA double-strand breaks. Genetics, 2010, 186: 757–761

[66] Lacombe S, Rougon-Cardoso A, Sherwood E, Peeters N, Dahl-beck D, van Esse H P, Smoker M, Rallapalli G, Thomma B, Staskawicz B, Jones J, Zipfel C. Interfamily transfer of a plant pattern-recognition receptor confers broad-spectrum bacterial re-sistance. Nat Biotechnol, 2010, 28: 365–369

[67] Römer P, Recht S, Lahaye T. A single plant resistance gene pro-moter engineered to recognize multiple TAL effectors from dis-parate pathogens. Proc Natl Acad Sci USA, 2009, 106: 20526– 20531

[68] Zou L-F(邹丽芳), Chen G-Y(陈功友), Wu X-M(武晓敏), Wang

J-S(王金生). Cloning and analysis of diverse members of avrBs3/PthA Family of Xanthomonas oryzae pv. oryzicola. Sci Agric Sin (中国农业科学), 2005, 38(5): 929–935 (in Chinese with English abstract)

[69] Zhao B, Lin X, Poland J, Trick H, Leach J, Hulbert S. A maize

resistance gene functions against bacterial streak disease in rice. Proc Natl Acad Sci USA, 2005, 102: 15383–15388

[70] Tian D, Yin Z. Constitutive heterologous expression of avrXa27

in rice containing the R gene Xa27 confers enhanced resistance to compatible Xanthomonas oryzae strains. Mol Plant Pathol, 2009, 10: 29–39