水稻与稻瘟病菌互作的分子机制研究进展
2018-01-18陈其国韦淑亚章萍
陈其国 韦淑亚 章萍
(1武汉职业技术学院生物工程学院,武汉430074;2浙江省杭州市富阳区委农业和农村工作办公室,杭州311400;第一作者:chenqiguo1008@126.com)
真菌Magnaporthe grisea引起的稻瘟病是最具毁灭性的水稻病害之一,严重影响水稻生产和粮食安全,通过选育和利用抗病品种来防控稻瘟病是既经济又有效的手段[1]。然而,由于稻瘟病菌毒性小种的不断变异,迫使水稻育种在探索持久抗性的同时,采用多基因聚合和基因轮换等利用抗病基因的策略。
随着水稻和稻瘟病菌全基因组测序计划的完成[2-3],从分子水平上认识水稻与稻瘟病菌的互作机制已成为可能。深入解析病原菌效应分子、无毒基因-抗性基因的互作及水稻应答稻瘟病菌入侵的信号调控网络等,对通过遗传育种和基因工程等手段改良品种抗病性,从而保障水稻安全生产具有十分重要的意义。
1 稻瘟病菌激发子
激发子(elicitor)是指参与诱导植物产生防卫反应的一类信号物质,包括来源于病原菌的外源性激发子以及植物自身产生的内源性激发子[4]。外源激发子也称为真激发子,是来自病原菌的信号分子,包括病原菌相关分子模式(pathogen-associated molecular patterns,PAMPs)和效应分子(effectors)。内源激发子是植物细胞间信号传导系统的组成部分,如病菌侵染过程中引起植物细胞壁降解得到的果胶寡聚物等。
稻瘟病菌激发子类型多样,如鞘脂、几丁质和小分子分泌蛋白等。有些分泌蛋白为无毒蛋白,和水稻中的抗病蛋白之间符合“基因对基因”假说,如AvrPiz-t是一个无毒基因,编码1个103氨基酸组成的分泌蛋白,能够直接被寄主细胞内的抗病基因Piz-t产物所识别,从而触发免疫反应[5]。
2 水稻抵抗稻瘟病的免疫应答反应
在与病原菌协同进化的过程中,水稻形成了复杂的防卫反应机制,进化出两层先天免疫系统来应对稻瘟病菌的侵染。第1层为病原菌相关模式分子诱发的免疫反应(PAMP-triggered immunity,PTI),第 2层为效应分子诱发的免疫反应(Effector-triggered immunity,ETI)[6-8]。这两种免疫反应诱导水稻产生抗病性,通常可分为3个步骤:第1步,信号感知,即水稻通过各种受体来识别病原物中的PAMPs或效应分子;第2步,信号经G蛋白、Ca2+流等传递并放大后,进一步激活丝裂原活化蛋白激酶和NADPH氧化酶,释放活性氧;第3步,诱导防卫基因表达,积累抗病原物的次级代谢产物,加厚细胞壁,侵入位点的细胞程序性死亡等[6-8]。
2.1 PAMPs诱导的PTI免疫反应
遭受稻瘟病菌侵染后,水稻表达的模式识别受体(pattern recognition receptors,PRR)可特异识别病原菌的PAMPs,从而激活对稻瘟病菌的防卫反应。这种由PRR识别PAMPs并诱导的防卫反应,是寄主的基础免疫反应,简称 PTI[7]。
几丁质是真菌细胞壁的重要组分,作为一种经典的PAMP,由其诱导的PTI研究得较为深入。研究表明,PTI诱导效应随几丁质聚合度的提高而增强,且聚合度小于5的几丁质短链不足以引起免疫反应[9]。糖蛋白OsCEBiP是一种分布于细胞膜上的模式识别受体,包含一个跨膜结构域和两个LysM结构域,可特异识别并结合几丁质寡聚糖[10-12]。除OsCEBiP外,细胞膜上还存在其他的几丁质识别受体,如LYP4和LYP6[13]。但仅有OsCEBiP或LYP4/6,不能将几丁质信号由胞外向胞内传输,受体激酶OsCERK1则可分别与这些受体结合形成复合体,完成信号的接力[11,14]。紧接着,OsCERK1通过胞质结构域磷酸化OsRacGEF1的C端S549,从而激活OsRacGEF1,而OsRacGEF1是鸟嘌呤核苷酸交换因子,能激活小GTP酶OsRac1[15]。因此,由OsCEBiP/OsCERK1-OsRacGEF1-OsRac1组成的模块构成了PTI免疫反应早期阶段的重要信号通路。OsRac1被激活后,将通过多种途径开启下游的防卫反应。其一,Os-Rac1通过激活NADPH氧化酶OsRbohB,迅速产生活性氧[16],并通过抑制ROS清除相关基因如OsMT2b的表达,确保ROS的积累[17]。其二,OsRac1通过调控NADPH氧化酶和肉桂酰辅酶A还原酶OsCCR1的活性,控制木质素的合成,而木质素是植物防卫反应中的重要因子,因为它形成了病原菌无法降解的机械壁垒[18]。其三,OsRac1通过介导丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)级联反应,诱导下游免疫应答。具体过程包括:OsRac1与OsMAPK3/6互作,并通过OsMKK4将其激活,激活后的OsMAPK3/6进入核内进一步磷酸化激活bHLH转录因子RAI1,随后RAI结合在靶标基因PAL和OsWRKY19的启动子区域,从而启动这2个防卫相关基因的表达,进而调控细胞程序化死亡、植保素合成和病程相关基因表达等进程[19-20]。
在上述免疫应答通路中,OsRac1有激活态(GTP结合型)和失活态(GDP结合型)两种构象,对信号的转导起着分子开关的作用。鸟苷酸交换因子OsSWAP70A[21]和OsRacGEF1[15],参与催化OsRac1由GDP结合态转变成GTP结合态,激活OsRac1蛋白。反之,Rho GTP酶激活蛋白SPIN6催化OsRac1由GTP结合态转变成GDP结合态,令其失活[22]。此外,OsRac1不是独自发挥功能,而是与其他蛋白如OsRAR1、HSP90和HSP70形成了一个或多个免疫复合体,共同参与信号的传递[23]。而OsRAR1又能够与OsSGT1直接互作,在免疫应答中发挥协同或拮抗作用[24]。RACK1A则与复合体中的OsRAR1、OsSGT1等直接互作,发挥支架蛋白作用[25]。进一步研究发现,Hop/Sti1-Hsp90分子伴侣复合体能促进PRRs成熟,并依赖Sar1途径将其从内质网运输到质膜上。质膜上,Hop/Sti1、Hsp90与OsRac1以复合体形式存在,可能起链接OsCERK1和OsRac1的作用[26]。
除了上述由OsRac1介导的MAPK通路外,最近又发现一条独立于OsRac1的MAPK激活通路。OsCEBiP识别几丁质信号后,导致OsCERK1磷酸化,激活OsCERK1磷酸化胞质激酶 OsRLCK185,OsRLCK185再与OsMAPKKKε互作并将其激活,活化的OsMAPKKKε再激活 OsMKK4/5,最后,OsMKK4/5激活OsMPK3/6,从而开启下游的免疫反应[27]。此外,OsRLCK176也能与OsCERK1互作,位于OsCERK1的下游[28],可能与OsRLCK185功能冗余。
2.2 效应分子诱导的ETI免疫反应
虽然水稻拥有PTI免疫系统,但很多情况下仍然遭受稻瘟病菌的侵染并感病,这是因为稻瘟病菌能分泌一些效应分子,抑制PAMPs诱导的PTI免疫反应[7-8,29-30]。然而,水稻也进化出基于R蛋白的第二道防线,能识别或感知病原菌的效应分子,启动ETI免疫反应。被相应R蛋白识别并克服的病原菌效应分子,也称为无毒蛋白。截至目前,水稻上鉴定的稻瘟病抗性基因已多达24个[31],从稻瘟病菌中鉴定的无毒基因也有13个,分别为 PWL1[32]、PWL2[33]、PWL2D[34]、AvrPita[35]、ACE1[36]、AvrPik[37]、AvrPii[37-38]、AvrPia[37,39]、Avr1-CO39[40]、AvrPib[41]、AvrPiz-t[5]、AvrPi9[42]和 AvrPi54[43]。
R蛋白通过直接或间接地与效应分子互作,从而感知病原菌入侵并诱导抗病反应。据报道,Pita/AvrPita[35]、Pikh-1/AvrPik[37]、Pia/AvrPia[39,44]、Pi-CO39/Avr1-CO39[40,44]和 Pi54/AvrPi54[43]等组合可直接发生互作。以 Pi54/AvrPi54为例,无毒基因AvrPi54位于稻瘟病菌的4号染色体,编码一个由153个氨基酸组成的分泌蛋白(N末端是19个氨基酸长的信号肽序列),能与抗性蛋白Pi54的富亮氨酸重复区发生物理互作[43]。相比之下,Pii/AvrPii[38]、Piz-t/AvrPiz-t[5]等组合不直接发生互作,而是需借助其他“助手”蛋白来完成相互识别。以Pii/AvrPii为例,无毒基因AvrPii编码70个氨基酸的小分泌蛋白,与OsExo70-F2和OsExo70-F3形成复合体,而OsExo70-F3作为一种“诱饵”能直接与无毒蛋白互作,一旦互作会立即被Pii识别并启动防卫反应[38]。还有种特殊的情况,即R蛋白虽能与无毒蛋白直接互作,但仍需要“助手”蛋白的参与,如Pib/AvrPib组合。作为防卫蛋白Pib监测靶标蛋白ABIP1的磷酸化,当效应分子AvrPib进入寄主体内时,与ABIP1结合并使其磷酸化,一旦监测到磷酸化,Pib作为防卫蛋白的能力便被激活,从而与AvrPib效应蛋白互作,启动防卫反应[41]。
2.3 稻瘟病菌维持毒性的机制
在病原菌-水稻的长期协同进化过程中,稻瘟病菌也拥有了一些维持毒性的机制。一种有效的措施就是避免PAMPs如几丁质被寄主识别,如效应分子Slp1可以与CEBiP竞争性结合几丁质寡糖,从而阻断其被CEBiP识别[45]。再如α-1,3-葡聚糖可加固稻瘟病菌细胞壁,防止被水稻的降解酶水解,从而阻断几丁质释放,延缓寄主的免疫应答[46]。
在PTI或ETI介导的免疫应答过程中,常常会释放大量活性氧攻击病原菌,而稻瘟病菌也有多种方式调节寄主细胞的氧化还原状态以保护自己。稻瘟病菌DES1基因编码一个富含丝氨酸、在丝状子囊菌中非常保守的蛋白,DES1通过抑制水稻细胞活性氧爆发来降低自身对氧化胁迫的敏感性,并阻止寄主防卫相关基因的表达,确保菌丝的顺利侵染[47]。类似地,稻瘟病菌MoHYR1基因编码谷胱甘肽过氧化物酶,能参与清除寄主细胞内活性氧,将水稻体内的活性氧维持在一个较低水平[48]。除活性氧外,由NO衍生的活性氮,也能削弱病原菌的侵染[49]。然而,稻瘟病菌NMO2基因编码的氮酸酯单加氧酶,能催化硝基烷烃的氧化脱氮,减轻硝基氧化胁迫带来的病原菌脂质硝化,并维持寄主体内氧化还原平衡,避免触发寄主的防卫反应[50]。
3 结语及展望
近10多年来,水稻与稻瘟病菌的互作机制研究取得了很大发展,已成为研究植物与病原菌互作的模式,如稻瘟病菌入侵全过程的动态监测,水稻和稻瘟病菌的全基因组测序和重测序,越来越多的抗性基因和无毒蛋白的发现等。但稻瘟病菌与水稻之间的对话乃是一场旷日持久的“军备竞赛”,新抗性基因的产生又必然促进病原菌毒性基因的新变异,反之亦然。不管是PTI基础免疫反应还是ETI高级防卫体系,都是极其复杂的互作网络,目前的研究已经拉开了揭示该网络的序幕。后续的基础研究,仍需要鉴定该网络中更多的效应蛋白和抗性基因,并揭示它们的互作机制。育种上,除了聚合多个抗性基因之外,挖掘并利用更多的广谱抗性基因可能是一个更好的途径。
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