葉德友，漆永紅，李敏權(quán)

(1.甘肅省農(nóng)業(yè)科學(xué)院蔬菜研究所，甘肅 蘭州 730070；2.甘肅省農(nóng)業(yè)科學(xué)院植物保護(hù)研究所，甘肅 蘭州 730070；3.甘肅省農(nóng)業(yè)科學(xué)院，甘肅 蘭州 730070)

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植物與線蟲互作的信號傳導(dǎo)及調(diào)控機(jī)制研究進(jìn)展

葉德友1*，漆永紅2，李敏權(quán)3

(1.甘肅省農(nóng)業(yè)科學(xué)院蔬菜研究所，甘肅 蘭州 730070；2.甘肅省農(nóng)業(yè)科學(xué)院植物保護(hù)研究所，甘肅 蘭州 730070；3.甘肅省農(nóng)業(yè)科學(xué)院，甘肅 蘭州 730070)

植物寄生線蟲嚴(yán)重危害農(nóng)業(yè)生產(chǎn)，對全球作物產(chǎn)量造成重大經(jīng)濟(jì)損失。植物對線蟲的抗病和感病性作為作物生產(chǎn)中的關(guān)鍵性影響因子，一直是作物遺傳育種學(xué)家研究的重要課題之一，探明植物對線蟲抗病和感病性的內(nèi)在機(jī)理對于指導(dǎo)作物抗線蟲育種具有重要的理論意義和實(shí)踐價值。本文綜述了影響植物對線蟲抗病和感病性的內(nèi)在因素，包括植物抗性基因或蛋白、激素合成與信號傳導(dǎo)以及線蟲脅迫過程中產(chǎn)生的活性氧等信號傳導(dǎo)。國內(nèi)外近年來的研究認(rèn)為，植物對線蟲的抗病或感病性取決于多種信號通路間的協(xié)調(diào)互作，各種與線蟲抗性相關(guān)的信號通路間的交互對話構(gòu)成了復(fù)雜的信號傳導(dǎo)網(wǎng)絡(luò)，多種轉(zhuǎn)錄因子與小RNAs通過轉(zhuǎn)錄、轉(zhuǎn)錄后以及翻譯參與了信號傳導(dǎo)網(wǎng)絡(luò)的精細(xì)調(diào)控，這一高效控制的信號傳導(dǎo)網(wǎng)絡(luò)決定了寄主植物對線蟲的抗病或感病性。這些研究成果將為深入闡明植物與線蟲互作的信號傳導(dǎo)和調(diào)控機(jī)制奠定基礎(chǔ)，從而為植物線蟲防控新策略的制定提供理論依據(jù)。

線蟲；抗性基因；激素；活性氧；小RNA

植物在生長環(huán)境中往往會受到真菌、細(xì)菌、病毒和線蟲等病原微生物的脅迫，這些病原微生物分泌效應(yīng)分子進(jìn)入植物細(xì)胞引起植物發(fā)病。植物能夠識別病原相關(guān)的分子模式(pathogen-associated molecular patterns, PAMPs)，病原利用該模式識別植物受體(pattern recognition receptors, PRRs)，從而引起模式觸發(fā)免疫(pattern-triggered immunity, PTI)。植物為應(yīng)對病原微生物進(jìn)化出了一些特異的抗性(resistance, R)蛋白，R蛋白能夠識別病原效應(yīng)子引起效應(yīng)子觸發(fā)的免疫反應(yīng)(effector-triggered immunity, ETI)[1]。R蛋白通常含有核苷酸結(jié)合位點(diǎn)(nucleotide-binding site, NBS)與胞外富亮氨酸重復(fù)結(jié)構(gòu)域(leucine-rich repeat, LRR)，NBS具有NTP酶活性，LRR與植物受體和病原效應(yīng)子的互作有關(guān)，當(dāng)與病原效應(yīng)子接觸時，NBS作為分子調(diào)節(jié)器激活下游信號傳導(dǎo)，R蛋白能夠直接識別病原效應(yīng)子，也可以通過其他輔助因子識別病原[2]。PTI或ETI反應(yīng)的激活增強(qiáng)了植物對病害的抗性，抑制了病原菌的生長，抗病性與感病性植物的主要區(qū)別就在于抗病性植物能夠適時地識別入侵微生物，并能夠快速有效地啟動防衛(wèi)反應(yīng)[3]。

植物線蟲是農(nóng)業(yè)生產(chǎn)中的一類重要病原微生物，危害農(nóng)作物生產(chǎn)的植物線蟲主要包括根結(jié)線蟲(Meloidogynespp., root-knot nematodes, RKN)和孢囊線蟲(Globodera和Heteroderaspp., cyst nematodes, CN)。這些線蟲與寄主作物進(jìn)化出了復(fù)雜的互作關(guān)系，在植物體內(nèi)形成了高度特化的線蟲取食位點(diǎn)(nematode feeding site, NFS)。NFS是線蟲侵染植物后在根系中形成的細(xì)胞核分裂而細(xì)胞質(zhì)未經(jīng)分裂的多核細(xì)胞，是線蟲賴以生存的營養(yǎng)來源，如RKN在根系中形成的巨型細(xì)胞(giant cells)[4]，而CN將初始取食細(xì)胞與相鄰細(xì)胞間的細(xì)胞壁打破融合形成合胞體(syncytia)[5]。線蟲通過口針從NFS中攝取養(yǎng)分，口針分泌的效應(yīng)物對于NFS的形成與維持至關(guān)重要，這些效應(yīng)蛋白作為PAMPs或致病效應(yīng)子促使線蟲寄生，或調(diào)節(jié)植物的防衛(wèi)反應(yīng)，或改變植物的生理[6]。線蟲分泌的效應(yīng)蛋白既可以進(jìn)入細(xì)胞質(zhì)，與細(xì)胞周期、細(xì)胞骨架和細(xì)胞代謝組分發(fā)生互作，也可以在胞外積累，降解植物細(xì)胞壁，改變細(xì)胞壁結(jié)構(gòu)[7]。

了解植物-線蟲互作中的信號傳導(dǎo)與調(diào)控機(jī)制對于確定控制植物與線蟲互作關(guān)系的生物進(jìn)程至關(guān)重要。本文對國內(nèi)外近年來在影響植物對線蟲的抗病性(R)和感病性(susceptibility, S)因素等方面取得的研究進(jìn)展進(jìn)行了綜述，包括參與植物防衛(wèi)反應(yīng)的信號分子及其傳導(dǎo)途徑，不同R基因介導(dǎo)的線蟲抗性及其信號傳導(dǎo)，植物應(yīng)答線蟲脅迫中的植物激素合成、信號傳導(dǎo)以及活性氧(reactive oxygen species, ROS)的產(chǎn)生及其信號傳導(dǎo)，小RNAs在植物-線蟲互作中的調(diào)控作用等，旨在為全面解析植物與線蟲互作的信號傳導(dǎo)和調(diào)控機(jī)制奠定基礎(chǔ)。

1　植物-線蟲互作中的R蛋白及信號傳導(dǎo)

植物中蘊(yùn)藏著大量的抗線蟲資源，現(xiàn)已在植物基因組中定位了許多抗線蟲基因[8]。Cai等[9]首次從甜菜(Betavulgaris)中克隆出抗線蟲基因Hs1pro-1，此后其他一些抗線蟲基因如Mi-1[10]、Gpa2[11]、HeroA[12]、Gro1-4[13]和Ma[14]陸續(xù)獲得克隆，這些基因均編碼胞內(nèi)NBS-LRR類R蛋白。前人將NBS-LRR類蛋白分為TIR-NBS-LRR和CC-NBS-LRR兩種類型[8,15]，研究發(fā)現(xiàn)蛋白質(zhì)白介素受體(toll-interleukin receptor, TIR)與卷環(huán)結(jié)構(gòu)(coiled-coil, CC)在免疫反應(yīng)的信號傳導(dǎo)中發(fā)揮關(guān)鍵性作用[2]。NBS-LRR蛋白作為病原受體，通過檢測病原效應(yīng)子引發(fā)以過敏反應(yīng)(hypersensitive response, HR)為主要特征的ETI類抗性反應(yīng)。目前已從HR中鑒定到了一些與植物NBS-LRR蛋白互作的線蟲效應(yīng)子，如PCN效應(yīng)蛋白RBP-1，含SPRY結(jié)構(gòu)域，與Gpa2蛋白互作[16]，MAP-1、Cg-1基因編碼的RKN效應(yīng)蛋白，可能與番茄Mi-1蛋白互作[17-18]。植物NBS-LRR基因介導(dǎo)的線蟲抗性往往伴隨著HR類細(xì)胞壞死[16,19]，可能還存在其他一些未知的與植物R蛋白互作的線蟲效應(yīng)子。因此，深入研究不同結(jié)構(gòu)域如何感知病原進(jìn)行信號傳導(dǎo)將有助于解析R基因介導(dǎo)的線蟲抗性中的信號傳導(dǎo)通路。

番茄(Solanumlycopersicom)Mi-1是目前研究得較為透徹的抗線蟲基因，其編碼的R蛋白參與了RKN抗性。Mi-1介導(dǎo)的RKN抗性依賴于病原識別和Mi-1蛋白中LRR結(jié)構(gòu)域介導(dǎo)的抗性信號傳導(dǎo)[20]以及與NBS結(jié)構(gòu)域相關(guān)的ATP酶活性介導(dǎo)的抗性信號傳導(dǎo)[21]?，F(xiàn)已證實(shí)，Mi-1蛋白N端在激活Mi-1蛋白時既有正調(diào)控作用也有負(fù)調(diào)控作用[22]，番茄RKN抗性需要Mi-1上游Rme1基因的參與[23]。通過病毒誘導(dǎo)的基因沉默(virus-induced gene silencing, VIGS)證實(shí)，番茄Mi-1介導(dǎo)的RKN抗性需要HSP90-1和SGT1的參與[24]。依據(jù)R蛋白介導(dǎo)的信號傳導(dǎo)模型[2]，Mi-1編碼的NBS-LRR蛋白與HSP90-1和SGT1蛋白結(jié)合構(gòu)成R蛋白信號復(fù)合物，通過檢測線蟲效應(yīng)子誘導(dǎo)的Rme1編碼蛋白的構(gòu)象變化激活下游信號傳導(dǎo)通路，而Rme1可能是Mi-1蛋白的互作因子和線蟲效應(yīng)子的直接靶標(biāo)[24]。Gpa2介導(dǎo)的馬鈴薯(Solanumtuberosum)孢囊線蟲(potato cyst nematodes, PCN)抗性需要RNA-GTP的參與，RNA-GTP可激活Gpa2輔因子蛋白質(zhì)2的活性[16]。

Rhg1和Rhg4是與大豆(Glycinemax)孢囊線蟲(soybean cyst nematodes, SCN)的R/S有關(guān)的2個數(shù)量性狀位點(diǎn)(quantitative trait loci, QTL)，不同于上述抗線蟲R基因，QTL編碼區(qū)的胞外LRR激酶類R蛋白可能調(diào)控SCN抗性[25]?，F(xiàn)已證實(shí)，Rhg1介導(dǎo)的SCN抗性由3個基因決定，即編碼氨基酸轉(zhuǎn)運(yùn)蛋白、α-SNAP蛋白和損傷誘導(dǎo)的結(jié)構(gòu)蛋白基因，SCN抗性可能與包含這3個基因31 kb重復(fù)序列的拷貝數(shù)有關(guān)，多拷貝產(chǎn)生抗性，而單拷貝則對SCN表現(xiàn)感病。此外，Rhg1內(nèi)存在的差異甲基化區(qū)域與SCN抗性有關(guān)，預(yù)測一些表觀遺傳因素可能在Rhg1介導(dǎo)的SCN抗性中發(fā)揮重要作用[26-27]。Rhg4介導(dǎo)的SCN抗性與編碼絲氨酸羥甲基轉(zhuǎn)移酶(serine hydroxymethyltransferase, SHMT)基因中的2個單核苷酸多態(tài)性有關(guān)[28]，SHMT可能通過調(diào)控葉酸碳代謝影響植物對線蟲的R/S反應(yīng)，這是由于葉酸缺失導(dǎo)致線蟲誘導(dǎo)的合胞體細(xì)胞發(fā)生降解，線蟲因饑餓而死，Suzuki等[25]研究認(rèn)為Rhg4位點(diǎn)鄰近的LRR基因調(diào)控SCN抗性。迄今，國內(nèi)外尚未從Rhg1與Rhg4位點(diǎn)分離獲得與R蛋白類似的基因產(chǎn)物，今后仍需開展相關(guān)研究進(jìn)一步解析Rhg1與Rhg4在SCN抗性中的確切作用。

2　植物-線蟲互作中的激素信號傳導(dǎo)

線蟲侵染植物后，多種植物激素參與激活PTI和ETI的下游反應(yīng)，從而誘導(dǎo)或抑制對線蟲的防衛(wèi)反應(yīng)。水楊酸(salicylic acid, SA)、茉莉酸(jasmonic acid, JA)、乙烯(ethylene, ET)等信號分子在植物應(yīng)答線蟲脅迫中發(fā)揮重要作用，一些激素還參與了受線蟲侵染的植物生長發(fā)育的調(diào)控，如生長素(auxin, AX)等。脅迫類激素SA、JA和ET主要通過PR基因以及其他抗性因子調(diào)控植物對線蟲的R/S，生長類激素AX則通過調(diào)控NFS的起始與發(fā)育影響線蟲的寄生。植物應(yīng)答線蟲脅迫中每一種激素的作用較為復(fù)雜，這不僅與植物和線蟲的物種特異性有關(guān)，還取決于線蟲侵染時間的差異。此外，不同的激素信號通路之間存在著錯綜復(fù)雜的互作關(guān)系。

2.1水楊酸

SA信號參與了R基因介導(dǎo)的線蟲防衛(wèi)反應(yīng)，SA信號在R基因HeroA[29]、Mi-1[30]與QTL基因Rhg1[31]介導(dǎo)的線蟲抗性中表達(dá)增強(qiáng)，攜帶R基因Mi-1和HeroA的番茄中過表達(dá)NahG(編碼SA羥化酶)，轉(zhuǎn)基因植株降低了對RKN和CN的抗性[29,32]。SA同源物苯并噻二唑(benzothiadiazole, BTH)能夠恢復(fù)轉(zhuǎn)NahG番茄(含Mi-1)的RKN抗性，而缺失Mi-1的感病植株則未能獲得RKN抗性[32]。擬南芥(Arabidopsisthaliana)R基因介導(dǎo)的抗性和基礎(chǔ)防衛(wèi)需要SA信號下游組分AtWRKY70的參與[33]。外源施用SA后發(fā)現(xiàn)，含Mi-1的番茄中WRKY70表達(dá)增強(qiáng)，沉默WRKY70則使Mi-1介導(dǎo)的RKN抗性降低[34]。WRKY轉(zhuǎn)錄因子參與了PTI與ETI中防衛(wèi)反應(yīng)的表達(dá)調(diào)控[33]，甜菜孢囊線蟲(beet cyst nematode, BCN,Heteroderaschachtii)侵染擬南芥后，其根系中WRKY6、WRKY11、WRKY17、WRKY33均下調(diào)表達(dá)，促進(jìn)了線蟲及其NFS的發(fā)育[35]。

除了R基因介導(dǎo)的抗性之外，參與SA生物合成、信號傳導(dǎo)以及SA響應(yīng)的相關(guān)基因促進(jìn)了植物對線蟲的基礎(chǔ)抗性。擬南芥異分支酸合成酶基因(iso-chorismate synthase,ICS)突變體和NahG轉(zhuǎn)基因品系增強(qiáng)了擬南芥對SCN的敏感性[36]，說明SCN抗性需要內(nèi)源SA的積累。ICS是SA合成中的關(guān)鍵酶，大豆中過表達(dá)SA甲基轉(zhuǎn)移酶(SA methyltransferase, SAMT)基因，其根系中ICS表達(dá)增強(qiáng)，大豆對SCN表現(xiàn)出抗性[37]。SAMT通過將SA轉(zhuǎn)換為甲基水楊酸(methyl salicylic acid, MeSA)調(diào)控SA水平[38]，MeSA作為移動性信號分子參與了植物系統(tǒng)獲得性抗性(systemic acquired resistance, SAR)[39]。PAD4位于SA信號通路上游，通過與病原敏感性增強(qiáng)子(enhanced disease susceptibility 1, EDS1)互作對病原脅迫作出應(yīng)答[38]。Atpad4突變增加了擬南芥對SCN的敏感性，過表達(dá)Atpad4的野生型大豆對RKN的抗性增強(qiáng)[40]。在大豆與SCN親和與非親和互作中，根系中EDS1轉(zhuǎn)錄水平增加[41]，表明SA信號通路上游組分參與了線蟲抗性。SA信號通路下游主要受制于SA受體病程相關(guān)非表達(dá)子基因(non-expressor of pathogensis related 1,NPR1)的調(diào)控[42]，擬南芥NPR1缺失突變體增強(qiáng)了對SCN的感病性，而npr1-1誘導(dǎo)型抑制子(suppressor ofnpr1-1 inducible,SNI1)缺失突變體則增強(qiáng)了SCN抗性[36]，通過轉(zhuǎn)基因在煙草(Nicotianatabacum)和大豆中表達(dá)AtNPR1分別獲得了RKN和SCN抗性[43-44]，大豆中過表達(dá)AtTGA2使得SCN在根系中的寄生受到抑制[45]。

PR是研究較為透徹的防衛(wèi)基因，PR的誘導(dǎo)表達(dá)可作為SA介導(dǎo)的抗性反應(yīng)激活的重要標(biāo)志[38]。將SCN效應(yīng)子10A06轉(zhuǎn)入擬南芥抑制PR-1、PR-2和PR-5的表達(dá)，轉(zhuǎn)基因植株增強(qiáng)了對SCN的敏感性[46]。SA防衛(wèi)反應(yīng)的信號中斷影響SCN的寄生，抑制PR-1和PR-5的表達(dá)發(fā)現(xiàn)SCN在大豆根系中建立了NFS，而過表達(dá)AtPR-5檢測到大豆根系中SCN的寄生受到抑制，表明SA響應(yīng)的防衛(wèi)基因參與了線蟲抗性[45,47-48]。對RKN敏感的玉米(Zeamays)lox3突變體中PR-1強(qiáng)烈誘導(dǎo)表達(dá)[49]，LOX3編碼9-脂氧合酶，9-脂氧合酶將脂肪酸氧化成脂氧合酶和JA[50]，lox3突變體增加了JA與ET響應(yīng)及其生物合成基因的表達(dá)水平[49]。與施用茉莉酸甲酯(methyl jasmonic acid, MeJA)相比，外源噴施SA同源物BTH僅能輕微增強(qiáng)水稻(Oryzasativa)RKN抗性[51]。因此，盡管SA在植物對線蟲的R/S中發(fā)揮關(guān)鍵性作用，其他一些激素及其參與的信號通路在植物應(yīng)答線蟲脅迫中的作用也不容忽視。

2.2茉莉酸

與SA合成和信號傳導(dǎo)不同，JA信號與植物對線蟲的敏感性有關(guān)。通過COI受體中斷JA信號，發(fā)現(xiàn)攜帶Mi-1的抗性番茄對RKN的抗性減弱[52-53]。Mi-1介導(dǎo)的RKN抗性中JA與SA信號相互拮抗，外源MeJA處理后SA誘導(dǎo)的WRKY70的表達(dá)受到抑制[54]?，F(xiàn)已證實(shí)JA受體coi-1突變體顯著降低了感病番茄根系中RKN的卵塊數(shù)目，對線蟲敏感的番茄中JA生物合成增加[49,52,55]。擬南芥lox4突變體中參與JA生物合成的基因誘導(dǎo)表達(dá)使植物對RKN更敏感，暗示JA積累與線蟲敏感性有關(guān)[55]，對RKN敏感的玉米lox3突變體中JA合成基因同樣誘導(dǎo)表達(dá)[49]，RKN侵染后LOX4與ZmLOX3表達(dá)增強(qiáng)。因此，JA生物合成可能正調(diào)控植物對線蟲的敏感性[49,55]。

蛋白酶抑制劑(protease inhibitors, PI)可能是JA誘導(dǎo)線蟲抗性的下游調(diào)控子，番茄根系中高量表達(dá)多半胱氨酸蛋白酶抑制子(multicystatin)類基因與PI基因時發(fā)現(xiàn)RKN的侵染受到抑制[56]。Mj-FAR-1是對RKN特異的脂肪酸和視黃醇(retinol)結(jié)合家族蛋白成員，番茄中超量表達(dá)Mj-FAR-1發(fā)現(xiàn)JA響應(yīng)的蛋白酶抑制子(Pin2)和γ-硫堇(thionin)編碼基因的表達(dá)受到抑制[57]，表明Mj-FAR-1可能負(fù)調(diào)控RKN抗性。JA和ET在水稻RKN抗性中的作用強(qiáng)于SA，外源MeJA和ET處理均可增強(qiáng)RKN抗性，這與抗性相關(guān)基因的高量表達(dá)有關(guān)，葉面噴施JA或ET生物合成抑制劑增加了水稻RKN敏感性[51]，RKN侵染后水稻根系和莖中參與JA、ET生物合成與信號傳導(dǎo)的基因表達(dá)受到抑制[58]。此外，JA是水稻RKN抗性中必不可少的信號分子，而ET介導(dǎo)的RKN抗性依賴于JA的生物合成。JA合成受阻的水稻突變體葉面噴施ET不影響RKN的侵染，但ET信號傳導(dǎo)受阻后JA誘導(dǎo)的防衛(wèi)反應(yīng)仍然發(fā)揮作用[51]。

2.3乙烯

ET及其信號傳導(dǎo)能夠影響RKN和CN的基礎(chǔ)抗性。外源ET處理大豆根系使大豆對SCN更敏感，而ET抑制劑1-甲基環(huán)丙烯(1-methycyclopropene, MCP)和2,5-降冰片二烯(2,5-norbornadiene, NBD)減少了大豆根系中SCN的侵染，表明ET負(fù)調(diào)控線蟲抗性[59]。擬南芥ET突變體eto1、eto2和eto3對CN表現(xiàn)高感[60]，但對RKN的抗性增強(qiáng)[61]，說明ET突變體在CN和RKN抗性中的作用相反。擬南芥ET受體突變體etr1和ET信號突變體ein2、ein3對CN的敏感性減弱[60]，編碼UDP-葡萄糖異構(gòu)酶的基因受ET信號基因EIN2和EIN3的負(fù)調(diào)控，而UDP-葡萄糖異構(gòu)酶能夠促進(jìn)CN抗性[62]。ET非敏感突變體etr1、ers2、ein4和番茄Nr正向調(diào)控ET信號基因ein2、ein3、ein5和ein7，導(dǎo)致RKN侵染數(shù)量增加，而負(fù)調(diào)控ET信號突變體ctr1使RKN的侵染數(shù)目減少。因此，ET生物合成及其信號傳導(dǎo)負(fù)調(diào)控CN抗性而正調(diào)控RKN抗性，但其具體的作用機(jī)理尚不明晰。

ET響應(yīng)的轉(zhuǎn)錄因子EREBPs在大豆與SCN的非親和性互作中誘導(dǎo)表達(dá)，而在親和性互作中其表達(dá)受到抑制[63]，大豆和擬南芥中過表達(dá)大豆GmEREBP1，根系中PR類基因誘導(dǎo)表達(dá)，但轉(zhuǎn)基因植株并未增強(qiáng)擬南芥對SCN的抗性[64]。在過表達(dá)GmEREBP1的轉(zhuǎn)基因植株中，除了ET誘導(dǎo)的GmPR2、GmPR3和AtPDF1.2基因表達(dá)增強(qiáng)之外，SA響應(yīng)的AtPR1、GmPR1和AtPR2基因以及JA響應(yīng)的GmPR3和AtPDF1.2同樣誘導(dǎo)表達(dá)[64]。這些研究表明，ET生物合成及其信號傳導(dǎo)在對不同的線蟲R/S中發(fā)揮不同的作用，因此，ET在植物線蟲抗性中可能具有多效性。一是由于ET合成和信號基因突變體對CN和RKN的反應(yīng)不同，不同的線蟲種類與其寄主之間可能存在獨(dú)特的作用機(jī)制；另外，GmEREBP1轉(zhuǎn)基因植株中不同種類的PR蛋白得以誘導(dǎo)表達(dá)，說明線蟲侵染植物過程中，ET、SA和JA信號通路之間發(fā)生了復(fù)雜的交互對話。目前還很難確定ET在線蟲侵染過程中的確切作用，但可以肯定，ET在一些線蟲的致病性中發(fā)揮重要作用，可能間接影響線蟲抗性。

2.4生長素

線蟲侵染過程中AX的極性運(yùn)輸影響其在取食細(xì)胞中的分布。在CN侵染的初始階段，LAX3/AUX1增加AX輸入而PIN1減少AX輸出，使AX在侵染部位積累，而當(dāng)合胞體細(xì)胞擴(kuò)張時，PIN3和PIN4通過橫向運(yùn)輸將AX輸送至初始合胞體周圍細(xì)胞[65-66]。LAX3是線蟲分泌蛋白Hs19C07的直接靶標(biāo)，Hs19C07與LAX3結(jié)合激活了LAX3，促進(jìn)了合胞體的發(fā)育[66]。AX非敏感突變體對CN抗性強(qiáng)于其野生型，CN建立的NFS及其相鄰細(xì)胞中的AX水平瞬間增加[67-68]。在順式作用元件NtCel7中發(fā)現(xiàn)了AX響應(yīng)元件，NtCel7編碼煙草β-1,4-內(nèi)葡聚糖酶降解植物細(xì)胞壁，在RKN與CN取食細(xì)胞中強(qiáng)烈誘導(dǎo)表達(dá)[69]。上述研究表明，線蟲侵染后取食細(xì)胞中AX的局部與瞬時積累促進(jìn)了線蟲NFS的建立與線蟲寄生。

AX對線蟲R/S的影響通過AX響應(yīng)因子ARFs來實(shí)現(xiàn)，ARFs能夠激活或抑制AX響應(yīng)基因的表達(dá)[70]。BCN侵染擬南芥后ARFs基因家族表現(xiàn)出明顯的差異表達(dá)，AX積累和ARFs表達(dá)在NFS起始與早期發(fā)育中瞬間增加[68]，成熟合胞體和AX誘導(dǎo)的成熟根結(jié)中ARFs持續(xù)高量表達(dá)[71]，說明AX和ARFs在成熟NFS中發(fā)揮重要作用[72]。ARFs下游的AX響應(yīng)基因LBD16受RKN侵染后激活，暗示其在根結(jié)和側(cè)根中誘導(dǎo)表達(dá)，從而建立了根結(jié)形成與側(cè)根發(fā)育間的分子聯(lián)系[72]。ARFs是一些小RNAs和干擾RNAs的直接調(diào)控靶標(biāo)，ARFs對于NFS的生長發(fā)育具有明顯的促進(jìn)作用。NFS的啟動與形態(tài)建成依賴于AX，BCN侵染后ARFs下游的AtWRKY23在早期的合胞體發(fā)育中誘導(dǎo)表達(dá)，但與AX無關(guān)[73]，表明其他一些信號分子參與調(diào)控植物對線蟲早期侵染的應(yīng)答。AX通常與ET協(xié)同互作，但ET介導(dǎo)的線蟲敏感性與AX無關(guān)[61-62]，這種侵染和非侵染植株信號通路中AX依賴性的不一致，說明線蟲分泌的效應(yīng)子可能通過植物AX信號調(diào)控植物響應(yīng)[66]，線蟲分泌物中AX類物質(zhì)也可能在植物對線蟲的R/S中發(fā)揮調(diào)控作用[6-7]。

3　植物-線蟲互作中的ROS及信號傳導(dǎo)

植物在光合作用與呼吸作用中通常會產(chǎn)生ROS，ROS是含氧的化學(xué)分子，包括單線態(tài)氧(singlet oxygen, 1O2)、超氧化物(superoxide, O2-)、過氧化氫(hydrogen peroxide, H2O2)和羥基自由基(hydroxyl radical, ·OH)。ROS的產(chǎn)生與清除機(jī)制競爭性協(xié)調(diào)使體內(nèi)ROS水平保持相對穩(wěn)定，ROS的過量積累通常會導(dǎo)致脂類、蛋白質(zhì)和DNA等的氧化，進(jìn)而引起細(xì)胞壞死；植物遭受病原物侵染后，體內(nèi)ROS含量迅速增加引起氧化迸發(fā)，導(dǎo)致局部細(xì)胞壞死[74]。病原侵染后局部迸發(fā)的ROS能夠通過細(xì)胞間的傳輸系統(tǒng)傳遞，也可以和其他信號傳導(dǎo)通路結(jié)合產(chǎn)生SAR反應(yīng)。ROS的產(chǎn)生通常與植物防衛(wèi)反應(yīng)有關(guān)，質(zhì)外體中ROS的快速積累往往會導(dǎo)致HR類細(xì)胞壞死，從而限制了病斑的進(jìn)一步蔓延，引起抗病反應(yīng)[75]。

RKN能夠侵入抗、感番茄的根系中，但在侵染后48 h，攜帶Mi-1的抗線蟲番茄根系中的RKN侵入數(shù)目顯著少于感病番茄[76]。線蟲侵染后12 h，在親和與非親和互作的根系侵染部位均檢測到了氧化迸發(fā)，但是在非親和反應(yīng)(攜帶Mi-1的抗線蟲番茄)中，氧化迸發(fā)持續(xù)延長至侵染后48 h，同時出現(xiàn)明顯的細(xì)胞壞死[76]。NADPH氧化酶是植物與病原非親和反應(yīng)中ROS產(chǎn)生的主要來源，通過亞細(xì)胞定位在非親和反應(yīng)的HR中檢測到了H2O2[76]。RKN侵染后ROS迅速積累，可作為監(jiān)控細(xì)胞代謝變化最靈敏的信號之一，ROS積累的時空差異對于確定RKN促使寄主植物的發(fā)病進(jìn)程至關(guān)重要。質(zhì)外體中ROS的快速積累與植物抗病性有關(guān)[74-75]，編碼ROS清除酶(如過氧化物酶B)的RKN基因在線蟲寄生階段大量轉(zhuǎn)錄，保護(hù)RKN免受寄主植物氧化損傷，將這些RKN基因敲除導(dǎo)致RKN寄生數(shù)目減少[77]。最新研究表明，ROS負(fù)調(diào)控細(xì)胞壞死與擬南芥BCN抗性，RbohD和RbohF是ROS產(chǎn)生中必需的編碼NADPH氧化酶的2個基因，RbohD和RbohF缺失突變體在BCN侵染24 h后BCN寄生數(shù)目減少，合胞體體積減小，細(xì)胞壞死增多[78]。此外，RbohD和RbohF缺失突變體中細(xì)胞壞死的抑制與SA的積累無關(guān)，RbohD/RbohF雙突變體中SA響應(yīng)基因誘導(dǎo)表達(dá)，而過表達(dá)RbohD的轉(zhuǎn)基因植株中SA響應(yīng)基因的表達(dá)受到抑制，表明SA與ROS相互拮抗[78]。新發(fā)現(xiàn)的ROS能夠抑制細(xì)胞死亡和促進(jìn)BCN寄生，表明HR類細(xì)胞壞死的激活可能存在其他未知的信號傳導(dǎo)機(jī)理[79]，這進(jìn)一步說明ROS在植物對線蟲的R/S中具有不同的作用。

4　sRNAs在植物-線蟲互作中發(fā)揮調(diào)控作用

sRNAs(20-24nt)是在表觀遺傳、轉(zhuǎn)錄、轉(zhuǎn)錄后以及翻譯水平對植物防衛(wèi)反應(yīng)具有重要調(diào)控作用的小分子物質(zhì)，植物sRNAs主要包括小RNAs(miRNAs)和短干擾RNAs(siRNAs)[80]。miRNAs和siRNAs源于通過DICER類蛋白對雙鏈RNA前體的加工，產(chǎn)生的miRNAs和siRNAs裝入AGO蛋白形成RNA誘導(dǎo)沉默復(fù)合體，該復(fù)合體可與靶RNAs和DNAs結(jié)合[81]。植物miRNAs和siRNAs在生物脅迫中發(fā)揮重要作用，miRNAs和siRNAs調(diào)控參與重要生物過程的基因表達(dá)，如ROS的產(chǎn)生及其信號傳導(dǎo)[82]、激素信號傳導(dǎo)[83]以及PTI和ETI[80]等。

sRNAs在病程發(fā)生的調(diào)控網(wǎng)絡(luò)體系中處于核心地位。RKN侵染番茄與水稻根系后，與miRNAs、siRNAs合成和生物功能相關(guān)蛋白的編碼基因，包括DCLs、AGOs和RDRs，編碼DNA甲基化酶的基因以及組蛋白甲基化和脫乙酰的相關(guān)基因表現(xiàn)差異表達(dá)[48,84]，DNA、組蛋白甲基化和乙?；墙閷?dǎo)植物表觀遺傳基因表達(dá)的重要調(diào)控機(jī)制[85]，說明miRNAs和siRNAs的生物合成及其功能在植物應(yīng)答線蟲脅迫中發(fā)揮重要作用。利用擬南芥單、雙和三突變體檢測到了SCN反應(yīng)，檢測到的基因包括DCLs、RDRs以及參與合成miRNAs和siRNAs的多種異構(gòu)體，產(chǎn)生sRNAs的基因突變與野生型相比均降低了SCN敏感性[86]。差異表達(dá)miRNAs和siRNAs的預(yù)測靶標(biāo)表明sRNAs在植物線蟲病害發(fā)生中具有特定作用。R蛋白、ARFs、熱激蛋白(heat shock proteins, HSP)和Cu/Zn超氧化物歧化酶的編碼基因，以及各類轉(zhuǎn)錄因子均可作為一個或多個差異表達(dá)miRNAs和siRNAs的調(diào)控靶標(biāo)。擬南芥miR396在SCN侵染4 d后下調(diào)表達(dá)而7 d后上調(diào)表達(dá)，miR396靶標(biāo)生長調(diào)控因子(growth regulating factors, GRF)的表達(dá)則在SCN侵染后表現(xiàn)出與miR396相反的變化趨勢[86]。擬南芥GRF缺失突變體和過表達(dá)miR396的轉(zhuǎn)基因植株均降低了SCN感病性，過表達(dá)miR396的擬南芥根系中合胞體與SCN的侵染數(shù)目均減少，miR396結(jié)合位點(diǎn)缺失突變體中同樣檢測到了類似的特征[87]。GRF基因家族在細(xì)胞增殖與細(xì)胞體積變化中發(fā)揮正調(diào)控作用，miR396及其靶標(biāo)基因GRF對于SCN侵染的合胞體的發(fā)育至關(guān)重要。此外，合胞體中差異表達(dá)的近半數(shù)基因與擬南芥GRF缺失和miR396抗性突變體中差異表達(dá)的基因相同，表明在SCN誘導(dǎo)的合胞體中，miR396/GRF是基因表達(dá)重編程必不可少的調(diào)控體系[87]。棉花(Gossypiumhirsutum)受腎形線蟲(Rotylenchulusreniformis, reniform nematodes, RN)侵染后sRNAs的表達(dá)發(fā)生改變，一些miRNAs和siRNAs，如miR396和miR482在對RN存在抗、感差異的不同棉花基因型中表現(xiàn)出不同的表達(dá)模式。sRNAs的靶標(biāo)基因源于差異表達(dá)的sRNAs，包括植物免疫、激素信號、ROS的產(chǎn)生及信號傳導(dǎo)、sRNAs生物合成及其功能以及表觀遺傳調(diào)控基因，這進(jìn)一步說明sRNAs對上述信號通路具有調(diào)控作用。

一些NBS-LRR基因能夠從其mRNA轉(zhuǎn)錄本中逐級產(chǎn)生成簇的次生siRNAs，次生siRNAs的產(chǎn)生必需miRNA靶標(biāo)[88-90]。此外，一些次生siRNAs能夠?qū)⑵渌恍┓佬l(wèi)基因作為靶標(biāo)[89]，其余的siRNAs則參與了根系發(fā)育的AX信號調(diào)控[91-92]。NBS-LRR類蛋白與AX信號在植物對線蟲的R/S中具有明顯作用，推測miRNA/siRNA信號可能在整合線蟲信號系統(tǒng)中發(fā)揮重要作用。棉花sRNAs調(diào)控網(wǎng)絡(luò)涉及RN信號傳導(dǎo)，預(yù)測對RN響應(yīng)的miR482家族靶向NBS-LRR蛋白的編碼mRNA可以裂解為一族次生siRNAs，這些siRNAs同樣會將一系列的轉(zhuǎn)錄因子以及其他蛋白作為靶標(biāo)，其中許多靶標(biāo)是參與信號傳導(dǎo)的致病相關(guān)基因，從而為sRNA調(diào)控網(wǎng)絡(luò)通過產(chǎn)生次生siRNAs、編碼miRNAs和NBS-LRR蛋白的基因參與棉花和RN互作提供了初步證據(jù)，這在其他病原侵染的植物免疫反應(yīng)中已有過詳細(xì)的研究報道[88-89,93]。上述研究表明，miRNA通過形成次生siRNAs調(diào)控NBS-LRR的基因表達(dá)，從而在植物線蟲病害發(fā)生中發(fā)揮重要作用，但有關(guān)這一調(diào)控網(wǎng)絡(luò)的確切作用目前尚不明晰。寄主植物能夠進(jìn)行自主防衛(wèi)，可能通過下調(diào)特定miRNAs的表達(dá)，進(jìn)而引起參與線蟲抗性R基因的表達(dá)發(fā)生改變。通過sRNA抑制NBS-LRR的基因表達(dá)可作為植物自身的保護(hù)機(jī)制，這是因?yàn)镹BS-LRR轉(zhuǎn)錄及其蛋白水平的增加能夠引發(fā)植物HR或細(xì)胞壞死[94]。線蟲分泌的特異效應(yīng)子也可能通過誘導(dǎo)sRNA調(diào)控網(wǎng)絡(luò)，抑制了NBS-LRR的基因表達(dá)，促進(jìn)了線蟲的寄生，可能是由于植物與線蟲間的sRNAs傳遞抑制了線蟲致病性及其發(fā)育必需的基因表達(dá)[95-96]，線蟲分泌的效應(yīng)RNAs啟動了植物sRNA調(diào)控網(wǎng)絡(luò)。

5　小結(jié)

植物R蛋白識別線蟲分泌的效應(yīng)物引發(fā)了植物線蟲抗性，植物激素不僅在R基因介導(dǎo)的線蟲抗性中發(fā)揮重要作用，而且在植物基礎(chǔ)防衛(wèi)反應(yīng)中具有促進(jìn)作用，每一種激素在特定的植物-線蟲互作中具有不同的作用，ROS在R基因介導(dǎo)的非親和互作的線蟲寄生中發(fā)揮關(guān)鍵性作用，ROS的局部迸發(fā)與R基因介導(dǎo)的線蟲抗性中的HR間存在著必然聯(lián)系?？梢姡鞣N不同的信號分子在調(diào)控網(wǎng)絡(luò)體系中彼此間協(xié)同作用，并非獨(dú)立發(fā)揮其功能。線蟲效應(yīng)子與植物因子間的互作影響信號網(wǎng)絡(luò)的調(diào)控行為，在植物應(yīng)答線蟲脅迫中同樣發(fā)揮重要作用。sRNAs是病原抗性的重要調(diào)控子，參與了植物防衛(wèi)反應(yīng)中關(guān)鍵節(jié)點(diǎn)基因的調(diào)控，sRNAs可能在植物對線蟲R/S決定中處于核心地位，但仍需通過研究進(jìn)一步揭示和證實(shí)sRNAs在不同植物-線蟲互作中發(fā)揮的具體作用。高通量二代測序技術(shù)與網(wǎng)絡(luò)分析策略的應(yīng)用，不僅有助于發(fā)現(xiàn)植物對線蟲R/S中的關(guān)鍵信號通路，而且對于揭示信號通路間的交互對話機(jī)制與信號通路整合也將發(fā)揮重要作用。

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Research progress on signal transduction and regulation mechanisms in plant-nematode interactions

YE De-You1*, QI Yong-Hong2, LI Min-Quan3

1.Institute of Vegetables, Gansu Academy of Agricultural Sciences, Lanzhou 730070, China; 2.Institute of Plant Protection, Gansu Academy of Agricultural Sciences, Lanzhou 730070, China; 3.Gansu Academy of Agricultural Sciences, Lanzhou 730070, China

Plant parasitic nematodes pose a serious threat to agricultural production and result in significant economic losses in crops worldwide. A key factor in crop production is plant resistance or susceptibility to nematodes; therefore, this has been an important subject for researchers in the areas of crop genetics and breeding. Understanding the mechanisms of plant resistance or susceptibility to nematodes is of great theoretical significance and practical value to guide the breeding of nematode-resistant crops. In this paper, the mechanisms underlying plant resistance or susceptibility to nematodes are reviewed, including specific plant resistance genes or proteins, plant hormone synthesis and signaling pathways, and reactive oxygen signals that are generated in response to nematode attack. In recent years, many researchers have suggested that plant resistance or susceptibility to invading nematodes and nematode-secreted effectors is mainly determined by the coordination of different signaling pathways. Many studies have shown that crosstalk among various nematode resistance-related elements represents an integrated signaling network regulated by transcription factors and small RNAs at the transcriptional, posttranscriptional, and translational levels. Ultimately, the outcome of this highly controlled signaling network determines the resistance or susceptibility of the host plant to nematodes. These above-mentioned results lay the foundation for further research on the signal transduction and regulation mechanisms involved in the plant-nematode interaction, and thus, provide a theoretical basis for the development of new strategies to prevent and control plant nematodes.

nematodes; resistance genes; hormones; reactive oxygen species; small-RNA

10.11686/cyxb2015574

2015-12-23；改回日期：2016-01-27

國家自然科學(xué)基金項(xiàng)目(31560506)，農(nóng)業(yè)部西北地區(qū)蔬菜科學(xué)觀測實(shí)驗(yàn)站項(xiàng)目(2015-A2621-620321-G1203-066)，國家公益性行業(yè)(農(nóng)業(yè))科研專項(xiàng)(201503112-4)和甘肅省設(shè)施園藝作物高效栽培創(chuàng)新團(tuán)隊(duì)項(xiàng)目(2014GAAS02)資助。

葉德友(1972-)，男，甘肅民勤人，副研究員，博士。E-mail：ydy287@163.com

Corresponding author. E-mail：ydy287@163.com

http://cyxb.lzu.edu.cn

葉德友, 漆永紅, 李敏權(quán). 植物與線蟲互作的信號傳導(dǎo)及調(diào)控機(jī)制研究進(jìn)展. 草業(yè)學(xué)報, 2016, 25(10): 191-201.

YE De-You, QI Yong-Hong, LI Min-Quan. Research progress on signal transduction and regulation mechanisms in plant-nematode interactions. Acta Prataculturae Sinica, 2016, 25(10): 191-201.