楊玖霞, 張 浩, 王志龍, 王旭麗, 王國梁*

(1.湖南農(nóng)業(yè)大學(xué)農(nóng)學(xué)院, 長沙 410128;2. 中國農(nóng)業(yè)科學(xué)院植物保護(hù)研究所,植物病蟲害生物學(xué)國家重點實驗室, 北京 100193)

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E3泛素連接酶調(diào)控植物抗病分子機理研究進(jìn)展

楊玖霞1,2, 張 浩1,2, 王志龍1, 王旭麗2*, 王國梁1,2*

(1.湖南農(nóng)業(yè)大學(xué)農(nóng)學(xué)院, 長沙 410128;2. 中國農(nóng)業(yè)科學(xué)院植物保護(hù)研究所,植物病蟲害生物學(xué)國家重點實驗室, 北京 100193)

生物脅迫是影響我國農(nóng)作物生產(chǎn)的重要因素,也是當(dāng)前植物界研究方向涉及最為廣泛的領(lǐng)域之一。由泛素介導(dǎo)的降解途徑是生物體內(nèi)最為精細(xì)的調(diào)控體系,涉及對生物體的生長發(fā)育以及生物體對周圍環(huán)境適應(yīng)的調(diào)控等過程。E3泛素連接酶因?qū)Φ孜镉刑禺愋宰R別作用,被認(rèn)為是泛素化過程中最重要的組成部分。依據(jù)其結(jié)構(gòu)和功能的不同可以將E3泛素連接酶分為4個家族。越來越多的研究表明這些不同的E3家族成員可以參與植物抗病免疫反應(yīng)的各個過程。本文在簡要概括E3泛素連接酶分類的基礎(chǔ)上綜述了目前E3泛素連接酶參與調(diào)控植物抗病害方面研究進(jìn)展,并對今后研究方向進(jìn)行了展望,以期對抗病機理解析及抗病品種研發(fā)提供新思路。

UPS; 泛素化; E3泛素連接酶; PTI; ETI

植物與病原物在長期相互作用過程中協(xié)同進(jìn)化。寄主針對病原菌相關(guān)分子模式(pathogen-associated molecule pattern, PAMP)包括模式識別受體(pattern recognition receptor, PRR),誘導(dǎo)的免疫反應(yīng)PTI (PAMP-triggered immunity)[1]阻止病原物的侵染。為了抵御寄主的PTI,病菌進(jìn)一步分泌特異性的效應(yīng)蛋白分子(effector),而植物則進(jìn)化出專一的抗病(resistance, R)蛋白直接或間接識別病原菌特異擁有的效應(yīng)蛋白,產(chǎn)生效應(yīng)蛋白激活的免疫反應(yīng)(effector-triggered immunity, ETI)[2],以抵御病原物的侵襲,其中過敏反應(yīng)(hypersensitive response, HR)是最典型的一種表現(xiàn)[3]。植物PTI和ETI免疫反應(yīng)中存在很多交叉相似的信號途徑,比如蛋白質(zhì)的翻譯后修飾作用,JA(jasmonic acid)、SA(salicylic acid)、ET(ethylene)等信號分子的產(chǎn)生,活性氧和抗病相關(guān)基因的誘導(dǎo)。植物激素是系統(tǒng)性信號的重要組分,并且對植物防御病原物的抗性水平有極其重要的影響,植物與病原物相互作用的同時常伴隨著一些激素水平的明顯變化,已有大量試驗數(shù)據(jù)表明,這些變化及其相互之間的影響對植物抵抗生物脅迫至關(guān)重要[4]。

泛素/26S蛋白酶體(ubiquitin-26S proteasome system, UPS)途徑是真核生物細(xì)胞中一種重要的蛋白質(zhì)翻譯后修飾作用,涉及對生物體的生長發(fā)育以及生物體對周圍環(huán)境適應(yīng)性的精細(xì)調(diào)控[5-10]。該過程首先是靶蛋白被泛素分子(76個氨基酸所組成的多肽)修飾標(biāo)記,然后被蛋白酶體識別和降解。其中,泛素對靶蛋白的修飾過程是由泛素活化酶(ubiquitin-activating enzyme, E1)、泛素結(jié)合酶(ubiquitin-conjugating enzyme, E2)和泛素連接酶(ubiquitin-ligating enzyme, E3)3個酶所介導(dǎo)的級聯(lián)反應(yīng)共同催化完成[11]。E3泛素連接酶是一個種類繁多的大家族,主要負(fù)責(zé)對靶蛋白的特異性識別,進(jìn)而對其進(jìn)行泛素化修飾。近幾年,越來越多的研究證據(jù)表明E3泛素連接酶參與了各種抗病信號反應(yīng)途徑的調(diào)控。本文綜述了E3泛素連接酶在植物免疫反應(yīng)從最初病原物的識別到下游信號途徑等各個過程中所發(fā)揮的正調(diào)控或負(fù)調(diào)控作用。通過概括其參與植物抗病反應(yīng)的分子機理,增加對病原菌與植物分子互作機理的認(rèn)識,為重要農(nóng)作物分子育種提供新思路。

1 E3泛素連接酶分類

UPS是植物細(xì)胞內(nèi)蛋白質(zhì)降解的主要途徑,參與細(xì)胞內(nèi)80%以上蛋白質(zhì)的降解,與各種應(yīng)激反應(yīng)都有關(guān)聯(lián)[9,12]。Lee[13]和Mazzucotelli[14]等研究發(fā)現(xiàn)擬南芥中超過1 600個基因可以編碼UPS(Ub/26S proteasome system)相關(guān)功能蛋白(超過全基因組總數(shù)的6%),其中有多于1 400個基因編碼E3泛素連接酶;水稻中E3泛素連接酶基因也有1 300多個[15],這些龐大的數(shù)字說明了E3泛素連接酶在植物生命周期中的重要性。

E3泛素連接酶根據(jù)結(jié)構(gòu)的不同大致可以分為兩大類：單亞基類型和多亞基類型。其中前者又可分為HECT型(homologous to E6-associated protein C-terminus)、U-box型和RING型(really interesting new gene);常見的多亞基CRL復(fù)合體(Cullin-RING ligase)如SCF(Skp1, Cullin, F-box)類型連接酶[5,9,16]。HECT類型蛋白C端含有一個約350個氨基酸殘基結(jié)構(gòu)域,此結(jié)構(gòu)域包含有Ub-E2復(fù)合體的結(jié)合位點,其中保守的半胱氨酸位點可以用于接受Ub分子,在將底物泛素化之前首先以E3-Ub中間體形式存在[17]。而RING和U-box蛋白在結(jié)構(gòu)上是十分相似的單一多肽,他們可以通過鋅螯合物和氫鍵或者鹽橋?qū)⒎核胤肿?Ub)從Ub-E2中間復(fù)合物轉(zhuǎn)移到靶標(biāo)蛋白上[18-19]。RING結(jié)構(gòu)域主要包括兩種構(gòu)型,由半胱氨酸(C)和組氨酸(H)組成的8個氨基酸與鋅離子螯合而成的RING-H2和RING-HC構(gòu)型;這種類型的泛素連接酶除了獨立發(fā)揮作用以外,還可以CRL復(fù)合體的一個多亞基單位來行使功能。CRL形式的E3泛素連接酶是指由Cullin、RING-box和靶標(biāo)蛋白識別元件共同組成的一個復(fù)合體[9],常見的有SCF(Skp1, Cullin, F-box),BTB(bric-a-brac-tramtrack-broad complex),DDB(DNA damage-binding)[9]。其中DDB復(fù)合體可以調(diào)節(jié)DNA的修復(fù)、DNA復(fù)制以及轉(zhuǎn)錄,它可以被病毒所破壞。常見CRL復(fù)合體中,對底物的特異識別依賴于F-box蛋白,RING蛋白則與E2結(jié)合在一起[20]。這些不同類別的E3連接酶家族成員都先后被報道參與植物免疫反應(yīng)過程。比如,通過細(xì)胞膜上的模式識別分子(PRR)調(diào)控植物對病原分子相關(guān)分子模式的識別,并調(diào)節(jié)細(xì)胞內(nèi)的核苷酸綁定亮氨酸富集的免疫受體積累;另外E3泛素連接酶還可以一些囊泡運輸成分或核轉(zhuǎn)錄因子為靶蛋白來調(diào)控病原菌識別免疫反應(yīng)下游的各種信號途徑。

2 E3泛素連接酶和植物的抗病性

2.1 E3泛素連接酶調(diào)控植物PTI免疫過程

目前,已有不少例證發(fā)現(xiàn)E3泛素連接酶可作為調(diào)節(jié)子參與植物細(xì)胞質(zhì)膜PRRs(pattern-recognition receptors)識別病原菌PAMPs所介導(dǎo)的PTI免疫反應(yīng)過程(表1)。其中研究較為深入的有,Lu等在研究擬南芥PRR蛋白FLS2(flagellin-sensing 2)識別PAMP因子flg22所誘導(dǎo)的抗病信號反應(yīng)過程中發(fā)現(xiàn),在由受體蛋白FLS2和協(xié)同受體BAK1(brassinosteroid insensitive 1-associated kinase 1)形成復(fù)合體這一復(fù)雜的信號通路中,受flg22結(jié)合FLS2的誘導(dǎo),U-box泛素連接酶PUB12和PUB13(plant U-box)可被協(xié)同受體BAK1磷酸化,進(jìn)而引起PUB12和PUB13直接泛素化FLS2以促進(jìn)其降解,從而負(fù)調(diào)控FLS2介導(dǎo)的PTI免疫反應(yīng),抑制過量或持續(xù)的免疫反應(yīng)激活對植物所造成的傷害[21]。另一例發(fā)現(xiàn)為擬南芥中3個E3泛素連接酶PUB22、PUB23和PUB24的表達(dá)量受PAMP因子flg22和chitin以及病菌侵染的誘導(dǎo)表達(dá),同時,Pseudomonassyringaepv.tomato經(jīng)pub22/pub23/pub24突變體接種后,表現(xiàn)為對病菌的抗性增強、ROS含量增多、參與ROS形成相關(guān)基因RbohD表達(dá)量上調(diào)[22-23],以上結(jié)果表明這3個E3泛素連接酶負(fù)調(diào)控PAMPs誘導(dǎo)的PTI反應(yīng)。另外,擬南芥中ATL9基因編碼一個RING-finger結(jié)構(gòu)蛋白,具備E3泛素連接酶活性。表達(dá)模式分析發(fā)現(xiàn)ATL9基因的表達(dá)量與擬南芥防御白粉菌(Golovinomycescichoracearum)的基礎(chǔ)抗性呈正相關(guān)。chitin處理擬南芥后,檢測到ATL9受到明顯誘導(dǎo),這表明該基因可能直接參與擬南芥抵抗白粉菌的PTI反應(yīng)過程[24]。轉(zhuǎn)錄因子VpWRKY11可以激活JA相應(yīng)相關(guān)基因AOS(allene oxide synthase)和LOX2(lipoxygenase 2),負(fù)調(diào)控植物免疫反應(yīng)[53]。Yu等[39]在中國野葡萄(Vitispseudoreticulata)中發(fā)現(xiàn)RING型E3連接酶蛋白EIRP1通過降解VpWRKY11,正調(diào)控植物免疫。另外他們還發(fā)現(xiàn),在擬南芥中過表達(dá)EIRP1導(dǎo)致VpWRKY11、AOS和LOX2的基因表達(dá)下調(diào),并提高轉(zhuǎn)基因植株對細(xì)菌和真菌的抗性,上述研究揭示了E3連接酶介導(dǎo)的蛋白降解途徑在調(diào)控植物抗病相關(guān)的轉(zhuǎn)錄因子介導(dǎo)的抗病信號中具有重要作用。

表1 植物免疫信號途徑中參與調(diào)控作用的E3泛素連接酶

續(xù)表1 Table 1(Continued)

物種SpeciesE3泛素連接酶E3ligase類型Type靶蛋白Target免疫過程作用Signalpathway引文References水稻RiceAPIP6RINGAvrPiz-t正調(diào)控PTI[41]水稻RiceSPL11U-boxSPIN6負(fù)調(diào)控PTI[42-43]水稻RicePUB44U-boxXopPxoo正調(diào)控ETI[44]水稻RiceXB3RINGXA21正調(diào)控ETI[45]水稻RiceOsDRF1F-box未知正調(diào)控抗病反應(yīng)[46]煙草TobaccoCMPG1U-boxAVR3a正調(diào)控ETI[47]煙草TobaccoACRE276U-boxSRK1-likekinase正調(diào)控ETI[28,48]煙草TobaccoACRE74U-box未知正調(diào)控ETI[49]胡椒PepperCaRING1RING未知正調(diào)控HR反應(yīng)[50]油菜BrassicaARC1U-boxSRK2-likekinase正調(diào)控免疫反應(yīng)[51-52]

水稻EL5(riceN-acetylchitooligosaccharide elicitor-responsive gene)是一個RING-H2 finger型泛素連接酶,研究證明EL5和水稻E2結(jié)合酶基因OsUBC5b受到激發(fā)子N-乙酰幾丁寡糖誘導(dǎo)表達(dá),該結(jié)果表明EL5可能與OsUBC5b一起參與病原菌引發(fā)的水稻PTI抗病反應(yīng)[40]。在揭示水稻稻瘟病菌(Magnaportheoryzae)效應(yīng)因子AvrPiz-t和其相對應(yīng)的R基因Piz-t的相互作用機理的過程中,發(fā)現(xiàn)效應(yīng)子AvrPiz-t互作蛋白APIP6是一個RING型E3泛素連接酶,在APIP6干擾的水稻轉(zhuǎn)基因株系中發(fā)現(xiàn)PAMP誘導(dǎo)的活性氧含量降低,抗病相關(guān)基因表達(dá)下調(diào),對稻瘟病抗性明顯減弱。這表明E3泛素連接酶APIP6作為一個正調(diào)控因子參與水稻對稻瘟菌的PTI免疫過程[41]。另外,擬南芥PUB12/13在水稻中的同源蛋白SPL11被發(fā)現(xiàn)負(fù)調(diào)控植物細(xì)胞程序化死亡和基礎(chǔ)抗病性[42]。Liu等[43]最新研究發(fā)現(xiàn)SPL11通過泛素化降解Rho型小G蛋白激活酶SPIN6,從而調(diào)控小G蛋白OsRac1介導(dǎo)的防衛(wèi)反應(yīng)信號。

2.2 E3泛素連接酶調(diào)控植物ETI抗病反應(yīng)過程

病原菌為了抵御寄主的PTI免疫反應(yīng),進(jìn)一步分泌特異性效應(yīng)蛋白分子(effector),打破植物的第一道防線-PTI,而植物則進(jìn)化出專一的抗病蛋白(resistance protein, R protein)直接或間接識別病原菌特異擁有的效應(yīng)蛋白。目前為止，植物中已經(jīng)有近百個R基因被克隆,其中一些基因所涉及的抗病信號途徑已被證明與泛素化相關(guān)[54]。

2.2.1 E3泛素連接酶直接作用于Avr和R蛋白

已有例證表明,E3泛素連接酶可直接作用于Avr和R蛋白,將其泛素化并經(jīng)26S蛋白酶降解體系最終降解,從而達(dá)到對寄主R基因所介導(dǎo)的ETI抗病反應(yīng)的調(diào)控。在動物界中已有相關(guān)報道,沙門氏菌(Salmonella)效應(yīng)子SopE和SptP在病菌侵染過程的不同階段發(fā)揮各自的功能,但是研究發(fā)現(xiàn)哺乳動物Henle-407細(xì)胞可以通過體內(nèi)UPS將這兩個效應(yīng)子泛素化并降解,從而阻礙細(xì)菌的入侵[55]。近年來在植物中也發(fā)現(xiàn)有相似的調(diào)控過程,例如,擬南芥E3泛素連接酶CPR1可以與NB-LRR R蛋白SNC1和RPS2發(fā)生互作并將其泛素化,最終依賴UPS途徑降低這兩個抗病蛋白的積累,避免了植物免疫信號途徑過度激活帶給植物的過度耗能[25-26]。水稻中,OsPUB44已被研究發(fā)現(xiàn)可以與Xoo(Xanthomonasoryzaepv.oryzae)效應(yīng)子XopPxoo發(fā)生互作,將OsPUB44沉默后發(fā)現(xiàn)PGN或chitin引發(fā)的免疫反應(yīng)受到抑制,植物對Xoo防御反應(yīng)減弱,這表明OsPUB44在水稻中對ETI過程起正調(diào)控作用[44]。

2.2.2 E3泛素連接酶作用于HR反應(yīng)途徑

HR過敏反應(yīng)是寄主抗病蛋白在識別病菌效應(yīng)蛋白時所產(chǎn)生ETI反應(yīng)中的最典型的一種反應(yīng),研究發(fā)現(xiàn)E3泛素連接酶可通過調(diào)節(jié)HR反應(yīng)達(dá)到對ETI過程的調(diào)節(jié)。例如,擬南芥中NLR(LZ-NBS-LRR)抗性蛋白RPM1被發(fā)現(xiàn)接種丁香假單胞菌(P.syringae)后聚集在質(zhì)膜附近,但在HR反應(yīng)初期迅速消失[56]。深入研究發(fā)現(xiàn),兩個RING型E3連接酶蛋白RIN2和RIN3能夠與RPM1相互作用,表明泛素蛋白酶體降解途徑可能參與了RPM1介導(dǎo)的ETI抗病信號途徑。但rin2rin3雙突變體和野生型Col-0接種DC3000(AvrRpm1)后都可以檢測到RPM1的消失,表明RPM1的降解并不是由RIN2和RIN3直接引起的[27]。另外rin2rin3雙突變體植株也會發(fā)生HR反應(yīng),只是較野生型HR反應(yīng)弱,并且突變體植物中依然存在病原菌的生長,由此可以看出RIN2/RIN3 E3泛素連接酶在植物對抗P.syringae的反應(yīng)中可能只參與調(diào)控RPM1依賴的HR反應(yīng)中的某個分支。

Gilroy等人發(fā)現(xiàn)U-box類型的泛素連接酶CMPG1參與介導(dǎo)無毒基因和其相應(yīng)的抗性基因(Cf-9/Avr9, Cf-4/Avr4, Pto/AvrPto)之間的識別作用所誘導(dǎo)的寄主細(xì)胞壞死反應(yīng)[47],即該蛋白作為正向調(diào)節(jié)子促進(jìn)寄主對病菌的抗病反應(yīng),但其在HR反應(yīng)過程中自身會被UPS系統(tǒng)所降解。隨后研究發(fā)現(xiàn)致病疫霉(Phytophthorainfestans)效應(yīng)子AVR3a在活體營養(yǎng)階段可以與CMPG1發(fā)生互作,并改變CMPG1定位,以穩(wěn)定CMPG1的泛素連接酶活性,阻礙其被26S蛋白酶體途徑所降解;從而抑制INF-1所引發(fā)的細(xì)胞壞死抗病性反應(yīng)的產(chǎn)生[47]。有意思的是,煙草中ACRE74與荷蘭芹(Petroselinumcrispum)CMPG1[57]和擬南芥PUB20/PUB21高度同源,因而又被命名為NtCMPG1。功能研究發(fā)現(xiàn),在具有Cf9抗性基因背景的煙草中超表達(dá)NtCMPG1后,轉(zhuǎn)基因植株對含有無毒效應(yīng)子Avr9的菌株表現(xiàn)出更強的抗性并誘發(fā)植株強烈HR反應(yīng)。反之,該研究團(tuán)隊嘗試在番茄中干擾番茄同源基因CMPG1,結(jié)果發(fā)現(xiàn)番茄植株對黃枝孢霉(Cladosporiumfulvum)的抗性明顯降低[49]。同樣的,煙草中RNAi干擾ACRE276導(dǎo)致植株HR反應(yīng)消失。番茄中干擾ACRE276同源基因后,降低了含抗性基因Cf-9的植株的抗病性[28,48]。進(jìn)一步研究發(fā)現(xiàn),擬南芥中ACRE276同源基因PUB17也具有保守的功能,煙草中瞬時表達(dá)PUB17可以互補ACRE276干擾煙草的HR反應(yīng),而E3活性缺失的PUB17則不能互補該表型[28],表明U-box E3連接酶ACRE276和PUB17在調(diào)控ETI防御反應(yīng)中起著保守的重要作用。XA21是一個RLK(receptor-like kinase)蛋白,研究發(fā)現(xiàn)在水稻中RING-type E3泛素連接酶XB3可以與XA21發(fā)生互作,參與調(diào)控水稻對Xoo的抗性。同時XA21的積累需要XB3的存在,XB3能夠被XA21磷酸化。在煙草表皮細(xì)胞過表達(dá)XB3蛋白能夠引起細(xì)胞壞死現(xiàn)象,而細(xì)胞壞死現(xiàn)象的發(fā)生則依賴XB3的E3酶活性,這些結(jié)果表明泛素蛋白酶降解途徑參與了XA21介導(dǎo)的抗病信號途徑[45,58]。

2.2.3 E3泛素連接酶作用于調(diào)控抗病相關(guān)基因的轉(zhuǎn)錄因子

擬南芥中RING型E3泛素連接酶MIEL1與轉(zhuǎn)錄因子MYB30在細(xì)胞核中可發(fā)生互作,并導(dǎo)致MYB30的降解,從而降低MYB30轉(zhuǎn)錄激活的抗病相關(guān)基因的表達(dá),抑制植物免疫反應(yīng)[29-30]。類似地,擬南芥中另一個RING型E3泛素連接酶BOI1(BOS1 interactor1)與R2R3型MYB轉(zhuǎn)錄因子BOS1(botrytis susceptible1)在植物細(xì)胞核中互作并可體外泛素化BOS1,從而負(fù)調(diào)控植物對病原菌的抗性。生物學(xué)功能研究表明,BOI1的RNAi植株對灰霉病的抗性減弱而且表現(xiàn)出更不耐鹽性。而在BOI1的RNAi植株中過表達(dá)BOS1會增加植物對活性氧的耐受性和灰霉病的抗性[31]。

2.3 E3泛素連接酶調(diào)控抗病相關(guān)信號途徑

水楊酸SA在調(diào)控活體營養(yǎng)或者半活體營養(yǎng)型病原菌抗性中發(fā)揮著重要的作用。CaRING1是來自胡椒的一種E3泛素連接酶,其蛋白序列中包含一個跨膜結(jié)構(gòu)域和一個C端RING結(jié)構(gòu)域,接種毒性辣椒瘡痂病菌(Xanthomonascampestrispv.vesicatoria)之后檢測到該基因表達(dá)受到誘導(dǎo)。功能研究發(fā)現(xiàn)病毒介導(dǎo)的CaRING基因沉默后,降低了植株的抗病性與抗病相關(guān)基因PR1的表達(dá),并伴隨親和性細(xì)胞死亡發(fā)生。同時,葉片中SA水平顯著下降[50]。另外,BAH1也是一個RING型E3連接酶,擬南芥bah1-D(benzoic acid hypersensitive1-Dominant)突變體在接種病原菌后或者用SA前體處理后都可以積累大量的SA,并產(chǎn)生依賴SA的局部細(xì)胞死亡,表明BAH1通過SA信號轉(zhuǎn)導(dǎo)途徑抑制植物免疫反應(yīng)[32]。

最近,Xie等[59]發(fā)現(xiàn)F-box類型E3泛素連接酶COI1參與JA信號途徑并調(diào)控植物抗病性。COI1作為JA信號途徑中一個重要的組成成分,也是SCF類型E3泛素連接酶中一個成員。最新研究發(fā)現(xiàn),茉莉酸ZIM結(jié)構(gòu)域(JAZ)蛋白是SCF-COI1調(diào)控的泛素化作用底物。當(dāng)SCF-COI1復(fù)合體識別茉莉酸-異亮氨酸信號以后,JAZ被SCF-COI1 E3復(fù)合體泛素化,并進(jìn)入26S蛋白酶體使其降解,受抑制的轉(zhuǎn)錄激活因子被重新激活,JA信號途徑相關(guān)基因開始轉(zhuǎn)錄、表達(dá)[33-34]。OsDRF1是水稻中一個F-box類型E3連接酶,ABA處理水稻后檢測到OsDRF1表達(dá)量上調(diào),在煙草中過表達(dá)OsDRF1,超表達(dá)植株表現(xiàn)出ABA敏感以及對病毒和細(xì)菌抗性的提高[46]。上述研究揭示了E3連接酶在抗病相關(guān)激素SA/JA/ABA信號途徑中發(fā)揮作用。

3 病原物效應(yīng)因子作為E3泛素連接酶參與調(diào)控植物防御反應(yīng)

E3泛素連接酶除了在植物抗病反應(yīng)中發(fā)揮著重要作用以外,新近研究表明病原菌相關(guān)效應(yīng)因子具有E3泛素連接酶的活性,以抑制寄主的PTI和ETI防衛(wèi)反應(yīng)。例如,番茄假單胞菌(P.syringaepv.tomato)無毒蛋白AvrPtoB是一個TypeⅢ效應(yīng)因子(T3E)[60]。AvrPtoB可以與番茄中的一個激酶Fen互作,以激活植物ETI反應(yīng)。然后,最近研究發(fā)現(xiàn)AvrPtoB的C端含有一個Ring/U-box結(jié)構(gòu)域[61],該結(jié)構(gòu)域具有E3泛素連接酶活性。且接種敲除該結(jié)構(gòu)域的AvrPtoB突變體菌株后,含F(xiàn)en基因的番茄植株表現(xiàn)出HR反應(yīng)[62]。在擬南芥中,AvrPtoB可通過其N端結(jié)構(gòu)域與模式識別受體FLS2及其共同受體BAK1在體內(nèi)發(fā)生相互作用,并催化FLS2的激酶結(jié)構(gòu)域被泛素化,以抑制植物PTI免疫過程[63-65];而在番茄中AvrPtoB可以特異性地泛素化Fen蛋白激酶,促進(jìn)Fen蛋白被26S蛋白酶降解體系降解,從而減弱番茄植株對假單胞菌的抗性,促進(jìn)病原菌的侵染和定殖[66]。

根瘤菌(Rhizobiumsp. strain NGR234)的效應(yīng)子蛋白NopM (nodulation outer protein M)編碼一個NEL(novel E3 ubiquitin ligase)類似E3蛋白,煙草中異源表達(dá)該基因后,植物中活性氧積累受到明顯抑制,防御反應(yīng)相關(guān)基因表達(dá)下調(diào)[67]。

最近的晶體結(jié)構(gòu)分析發(fā)現(xiàn),黃單胞菌效應(yīng)蛋白XopL編碼一種新型E3泛素鏈接酶,其在結(jié)構(gòu)上與沙門氏菌和志賀氏菌的Type III效應(yīng)蛋白高度同源。生物化學(xué)活性分析表明XopL能與植物E2結(jié)合酶發(fā)生特異互作并表現(xiàn)E3活性,這種E3泛素連接酶活性對細(xì)胞凋亡的誘導(dǎo)和植物免疫反應(yīng)的抑制起著關(guān)鍵作用[68]。

4 總結(jié)與展望

過去10年期間,科學(xué)家對泛素蛋白酶體系在植物生物學(xué)中的研究已延伸至各個方面,大量證據(jù)表明即使在單一的信號反應(yīng)途徑中UPS也有眾多的控制點,因此,一些特異的UPS系統(tǒng)組成元件比如E3泛素連接酶等的破壞、修飾或者重組都可以直接或者間接地對植物激素信號途徑、基因轉(zhuǎn)錄、形態(tài)發(fā)生、對周圍惡劣生長環(huán)境的抵御以及植物與病原物之間的競爭等過程的調(diào)控產(chǎn)生很大的阻力,從而影響到農(nóng)業(yè)生產(chǎn)。另外,泛素分子還可以通過3種酶的共同作用完成一些蛋白的正確組裝和折疊并參與一些蛋白的活性調(diào)節(jié)。盡管近期的遺傳學(xué)研究使人類對UPS的功能有了初步的認(rèn)識,但詳細(xì)的作用機理仍有待于探索。

植物中,所發(fā)現(xiàn)的編碼UPS相關(guān)蛋白的基因中,有很大部分都編碼E3泛素連接酶,研究E3與植物免疫的關(guān)聯(lián)有助于更好地理解植物與病原菌互作,但是有些E3泛素連接酶雖然發(fā)現(xiàn)其在宿主免疫抗病反應(yīng)中參與調(diào)控,但是其具體的分子機理尚不是很清楚。比如擬南芥PUB22、PUB23和PUB24雖然已經(jīng)證明可以抑制植物PTI免疫過程[22],但其中只有PUB22是通過UPS抑制植物免疫過程,而對于PUB23和PUB24,其具體的作用底物和所參與的上游信號途徑尚不清楚。故基于E3泛素連接酶基因在植物抗病反應(yīng)中所起的重要作用,一些問題的解決迫在眉睫,比如E3泛素連接酶的作用底物是什么？它是如何識別底物蛋白的？這些底物蛋白有何特點？解決了這些問題,不僅可以闡明植物對抗病原菌免疫反應(yīng)中錯綜復(fù)雜的互作關(guān)系,還可以增加人們對E3蛋白功能的認(rèn)識;更清楚地解釋植物通過自身免疫防御反應(yīng)對抗病原菌侵染機理,并為植物抗病新品種的改進(jìn)提供新思路。

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(責(zé)任編輯：田 喆)

Recent progresses in the regulation mechanism of E3 ligases in plant disease resistance

Yang Jiuxia1,2, Zhang Hao1,2, Wang Zhilong1, Wang Xuli2, Wang Guoliang1,2

(1. College of Agronomy, Hunan Agriculture University, Changsha 410128, China; 2. State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China)

Biotic stress is an important factor that affects crop production in China. It is also one of the most widely studied areas in plant sciences. Degradation mediated by the ubiquitin proteasome system (UPS) is one of the most sophisticated regulation systems in eukaryotes, which is involved in plant growth and development and in response to abiotic and biotic stresses. E3 ligase is considered as a key enzyme in the UPS due to its specific interactions with its substrates. Based on the differences in structure and function, E3 ligases can be divided into four main classes. In this paper, we review the recent progresses in the regulation mechanism of E3 ligases in plant disease resistance and propose new research directions.

UPS; ubiquitination; E3 ligase; PTI; ETI

專論與綜述Reviews

2015-02-09

2015-03-23

國家自然科學(xué)基金項目(31471737)

S 432.1

A

10.3969/j.issn.0529-1542.2015.04.001

* 通信作者 E-mail: lilywang0313@163.com；wang.620@osu.edu