何峰，張浩，劉金靈，王志龍，王國(guó)梁

1. 湖南農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，長(zhǎng)沙 410128；

2. 中國(guó)農(nóng)業(yè)科學(xué)院植物保護(hù)研究所，北京100193

水稻是最重要的糧食作物之一，是全球 50%左右人口的主食。稻瘟病是由子囊菌(Magnaporthe oryzae)引起的一種嚴(yán)重的水稻病害，為植物十大真菌性病害之一[1]。全球每年由于稻瘟病危害造成的產(chǎn)量損失可達(dá)水稻總產(chǎn)的 10%～30%[2]。近 10中(2004～2013)我國(guó)稻瘟病年平均危害面積在 8000萬(wàn)畝左右(數(shù)據(jù)來(lái)源于全國(guó)農(nóng)業(yè)技術(shù)推廣服務(wù)中心網(wǎng)站，http:// www.moa.gov.cn/sydw/njzx/)，水稻平均單產(chǎn)約430公斤/畝(數(shù)據(jù)來(lái)源于國(guó)家統(tǒng)計(jì)局網(wǎng)站，http://www.stats. gov.cn/)，按每畝減產(chǎn)5%計(jì)算，年造成產(chǎn)量損失超過(guò)15億公斤。盡管利用抗病品種是防治稻瘟病最經(jīng)濟(jì)有效的措施，但是由于田間稻瘟菌群體頻繁變異，往往導(dǎo)致抗病品種在應(yīng)用數(shù)年后就喪失抗性。此外，我國(guó)近年育成水稻品種整體抗性水平并不高。例如，2004～2008年國(guó)審 174個(gè)品種稻瘟病平均抗病指數(shù)為 5.5，屬于中感級(jí)別(數(shù)據(jù)來(lái)源于國(guó)家水稻數(shù)據(jù)中心網(wǎng)站，http://www.ricedata.cn/)。因此，發(fā)掘新抗病種質(zhì)資源并發(fā)展新的高效抗病育種技術(shù)一直是水稻抗病育種的迫切需求。

植物在與病原菌長(zhǎng)期協(xié)同進(jìn)化過(guò)程中形成了兩種天然免疫機(jī)制[3]，即病原菌相關(guān)分子模式(Pathogenassociated molecular patterns，PAMPs)誘導(dǎo)的抗病反應(yīng)機(jī)制(PAMP-triggered immunity，PTI)和病原菌效應(yīng)蛋白(Effector)誘導(dǎo)的抗病反應(yīng)機(jī)制(Effector-triggered immunity，ETI)。PTI通常由植物細(xì)胞表面模式識(shí)別受體(Pattern recognition receptors，PRRs)識(shí)別保守的病原菌PAMPs分子，然后激活植物體內(nèi)相對(duì)較弱的基礎(chǔ)防御反應(yīng)。ETI則依賴于植物抗病蛋白(R proteins)直接或間接識(shí)別病原菌分泌的效應(yīng)蛋白，進(jìn)而激發(fā)更為強(qiáng)烈的抗性反應(yīng)，以抑制病原菌侵染，通常表現(xiàn)為植物過(guò)敏反應(yīng)(Hypersensitive response，HR)。目前，植物PTI和ETI防衛(wèi)反應(yīng)分子機(jī)制在擬南芥(Arabidopsis thaliana)中已研究得比較深入[4]。

近年來(lái)，水稻與稻瘟菌互作已發(fā)展成植物與病原真菌分子互作研究的模式系統(tǒng)之一，人們?cè)谡J(rèn)識(shí)水稻稻瘟病PTI和ETI抗性機(jī)制及稻瘟菌致病性機(jī)制等方面取得了重要研究成果，并發(fā)展了一些具有重要應(yīng)用前景的育種新技術(shù)。本文總結(jié)了近年水稻抗稻瘟病PTI和ETI天然免疫分子機(jī)制研究的最新進(jìn)展，并探討了水稻抗病育種改良的新技術(shù)策略，同時(shí)對(duì)當(dāng)前水稻抗稻瘟病機(jī)制研究與抗病育種應(yīng)用的問(wèn)題和挑戰(zhàn)進(jìn)行了展望。

1 稻瘟病PTI防御機(jī)制

PTI是由PRR蛋白識(shí)別PAMPs而激發(fā)的抗性反應(yīng)。PAMPs是病原菌中一類保守的結(jié)構(gòu)性分子，如細(xì)菌鞭毛蛋白(Flagellin，flg22)、延伸因子(Elongation factor Tu，EF-Tu)、Ax21(Sulfated peptide Ax21)、肽聚糖(Peptidoglycan，PGN)、脂多糖(Lipopolysaccharides，LPS)、真菌細(xì)胞壁多糖、幾丁質(zhì)、葡聚糖等[5～8]。在擬南芥中，PTI信號(hào)激活分子機(jī)制已經(jīng)有了較系統(tǒng)地研究[9～11]。近年來(lái)，水稻 PTI信號(hào)響應(yīng)機(jī)制也取得了重要的進(jìn)展。

1.1 flg22與flg22受體OsFLS2及其介導(dǎo)的信號(hào)通路

flg22是細(xì)菌鞭毛蛋白 N 端一段含 22 個(gè)氨基酸的保守多肽，該肽段是鞭毛蛋白誘導(dǎo)植物抗性反應(yīng)的關(guān)鍵功能結(jié)構(gòu)[12]。擬南芥中，flg22能夠被PRR受體FLS2(Flagellinsensing 2)識(shí)別與結(jié)合，并激活下游抗病信號(hào)。FLS2編碼一個(gè)絲氨酸/蘇氨酸類受體蛋白激酶(Receptor-like kinase，RLK)。研究表明，水稻中FLS2同源基因OsFLS2也具有類似的保守功能。OsFLS2能夠與flg22結(jié)合，并能互補(bǔ)擬南芥fls2突變體的表型[13]。過(guò)表達(dá)OsFLS2增強(qiáng)了水稻對(duì)flg22的應(yīng)答，并激活了細(xì)胞死亡等抗性反應(yīng)[13]。

最近，晶體結(jié)構(gòu)研究發(fā)現(xiàn)flg22能夠引起FLS2和蛋白激酶BAK1(BRI1-associated receptor kinase 1)形成異源二聚體，并導(dǎo)致 FLS2和 BAK1相互磷酸化[14]。FLS2-BAK1二聚化后，通過(guò)磷酸化蛋白激酶BIK1(Botrytis-induced kinase 1)，而激活下游信號(hào)通路[15]。bik1突變體降低了擬南芥對(duì)flg22的敏感性，并增強(qiáng)了對(duì)假單胞菌 DC3000的感病性[15]。最新研究發(fā)現(xiàn)，BIK1通過(guò)磷酸化NADPH氧化酶RbohD，而誘導(dǎo)ROS的產(chǎn)生[16,17]。OsBAK1過(guò)表達(dá)水稻出現(xiàn)葉夾角小、發(fā)育不良和矮化等表型[18]。過(guò)表達(dá)OsBIK1增強(qiáng)了水稻對(duì)稻瘟病的抗性，且OsBIK1能夠互補(bǔ)擬南芥bik1突變體的功能[19]，但水稻中OsFLS2與 OsBAK1是否具有與擬南芥類似的保守功能，還有待深入研究。

1.2 幾丁質(zhì)與幾丁質(zhì)受體 OsCEBiP及其介導(dǎo)的信號(hào)通路

幾丁質(zhì)，即β-(1,4)-N-乙酰氨基-2-脫氧-D-葡聚糖，是真菌細(xì)胞壁的重要組分，也是誘導(dǎo)植物 PTI反應(yīng)的一類 PAMPs。擬南芥中，幾丁質(zhì)能夠被含LysM結(jié)構(gòu)域的類受體蛋白激酶CERK1(Chitin elicitor receptor kinase 1)識(shí)別、結(jié)合并激活 PTI反應(yīng)[20～22]。但不同的是，水稻中幾丁質(zhì)不能直接結(jié)合 CERK1的同源蛋白 OsCERK1，而是先與跨膜蛋白OsCEBiP(Chitin elicitor binding protein)結(jié)合，再由OsCEBiP與 OsCERK1形成異源二聚體，進(jìn)而傳導(dǎo)對(duì)幾丁質(zhì)的響應(yīng)，激活下游信號(hào)[23,24]。OsCEBiP也編碼含LysM結(jié)構(gòu)的類受體蛋白(RLP)，但無(wú)胞內(nèi)的激酶結(jié)構(gòu)域，為非典型的PRR蛋白。生物學(xué)功能分析表明，水稻OsCEBiP敲除突變體顯著抑制了幾丁質(zhì)誘導(dǎo)的 PTI反應(yīng)，并增強(qiáng)了對(duì)稻瘟病菌的感病性[23]。OsCERK1RNAi轉(zhuǎn)基因水稻也顯著抑制幾丁質(zhì)引起的防衛(wèi)反應(yīng)[24]。

最新研究發(fā)現(xiàn)，OsCERK1能夠磷酸化激活PRONE型鳥(niǎo)苷酸交換因子 OsRacGEF1，促進(jìn) Rho型小G蛋白OsRac1與GTP結(jié)合，變?yōu)榛钚誀顟B(tài)[25]。OsRac1為水稻PTI和ETI防御信號(hào)中一個(gè)關(guān)鍵性集成調(diào)控因子[26]?；钚詰B(tài)的 OsRac1能夠激活下游多樣性的抗病信號(hào)途徑。例如，OsRac1與NBS-LRR 蛋白Pit直接互作，激活ETI信號(hào)；OsRac1能夠激活膜上的呼吸爆發(fā)氧化酶 OsRbohB，氧化 NADPH，促進(jìn)ROS的產(chǎn)生。此外，OsRac1還能調(diào)節(jié)MAPK級(jí)聯(lián)反應(yīng)介導(dǎo)的抗病信號(hào)[25～27]。

1.3 幾丁質(zhì)、肽聚糖與受體蛋白LYP4和LYP6介導(dǎo)的抗病信號(hào)途徑

近來(lái)研究表明，水稻中OsCEBiP并不是幾丁質(zhì)的唯一受體，另外兩個(gè) LysM結(jié)構(gòu)域蛋白 LYP4和LYP6(Lysin motif-containing proteins)也參與了對(duì)幾丁質(zhì)的識(shí)別[28]。在細(xì)菌中LYPs識(shí)別細(xì)菌肽聚糖，而在植物中 LYPs能與肽聚糖相關(guān)的幾丁質(zhì)和結(jié)瘤因子(Nod factor)結(jié)合[29,30]。擬南芥中，LysM蛋白LYM1(At-LYP3)和 LYM3(At-LYP2)能夠識(shí)別結(jié)合肽聚糖而激活PTI反應(yīng)，但LYM1和LYM3是否也能識(shí)別幾丁質(zhì)，還有待進(jìn)一步研究[31～33]。而水稻 LYP4和LYP6具有雙重功能，能夠結(jié)合肽聚糖和幾丁質(zhì)。LYP4和LYP6是兩個(gè)細(xì)胞膜定位蛋白，具有N端的信號(hào)肽序列、2個(gè)LysMs結(jié)構(gòu)以及C端的糖基磷脂酰肌醇錨鉤。研究發(fā)現(xiàn)，細(xì)菌性病原菌及其他多種PAMPs，如肽聚糖、幾丁質(zhì)、脂多糖和flg22等，都可以誘導(dǎo)LYP4和LYP6的表達(dá)。生物學(xué)功能鑒定表明，LYP4或LYP6RNAi水稻均降低了肽聚糖或幾丁質(zhì)介導(dǎo)的防衛(wèi)反應(yīng)，并增強(qiáng)了對(duì)白葉枯菌(Xanthomonas oryzaepv.oryzae)和稻瘟菌的感病性[28]。

2 稻瘟病ETI防御機(jī)制

2.1 稻瘟病抗性基因與無(wú)毒基因的克隆

克隆抗稻瘟病基因與其對(duì)應(yīng)的無(wú)毒基因，并闡明它們之間的互作關(guān)系是認(rèn)識(shí)稻瘟病ETI免疫機(jī)制的關(guān)鍵。目前，水稻中已定位了超過(guò) 100個(gè)稻瘟病抗性基因，其中報(bào)道克隆了 22個(gè)(表 1)。這些已克隆的基因編碼包括核苷酸結(jié)合位點(diǎn)(Nucleotide binding sites，NBS)/富亮氨酸重復(fù)(Leucine rich repeat，LRR)蛋白(NBS-LRR)，如Pib、Pi-ta、Pi9、Pi2、Piz-t、Pi36、Pi37、Pikm、Pit、Pi5、Pid3、Pish、Pb1、Pi54、Pik、Pik-p、Pia、Pi25、Pi1和Pi35；受體蛋白激酶類(Receptor-like kinase protein，RLK)，如Pi-d2[34]；富脯氨酸類蛋白，如Pi21[35]等 3種類型的蛋白。此外，已克隆了 10個(gè)稻瘟菌無(wú)毒基因PWL1、PWL2、AvrPi-ta、AvrPiz-t、AvrPia、AvrPii、AvrPik/km/kp、Avr1-CO39、ACE1和AvrPi9(表 2)。

表1 已克隆的稻瘟病抗性基因(R)信息

表2 已克隆的稻瘟菌無(wú)毒基因(Avr)信息

2.2 R和Avr蛋白直接互作介導(dǎo)的ETI激活模式

在已克隆的R基因與Avr基因中，Pi-ta與Avr-Pi-ta[66]、Pik 與 AvrPik[67]、Pia 與 AvrPia[62]、PiCO39與Avr1-CO39[62]4對(duì)蛋白具有直接相互作用(圖1)。Pi-ta與AvrPi-ta最早被報(bào)道具有直接相互作用[66]。與Pi-ta/AvrPi-ta不同的是，Pik、Pia、PiCO39功能的實(shí)現(xiàn)需要兩個(gè)NBS-LRR蛋白共同作用，相應(yīng)的無(wú)毒蛋白 AvrPik、AvrPia、Avr1-CO39只與其中一個(gè)NBS-LRR成員互作。例如，Pik由 NBS-LRR基因Pik-1和Pik-2組成[49]，Pik-1被證明與AvrPik直接互作[67]。研究發(fā)現(xiàn)，Avr-Pik有Avr-Pik-A、B、C、D和E5個(gè)等位基因，同樣Pik也有 5個(gè)等位基因Pik*、Pikp、Pikm、Piks和Pikh。不同的Pik等位基因只識(shí)別特定的AvrPik等位基因。例如，Pikp只識(shí)別Avr-Pik-D，而不識(shí)別其他幾個(gè)等位基因，揭示了抗病基因與無(wú)毒基因之間的協(xié)同進(jìn)化機(jī)制[67]。此外，Pia由兩個(gè)反向串聯(lián)的NBS-LRR基因RGA4和RGA5組成[51]。AvrPia也只與 RGA5直接互作，且只與RGA5兩個(gè)轉(zhuǎn)錄本RGA5-A和RGA5-B中的RGA5-A互作[62]。RGA4和RGA5也被證明能夠識(shí)別無(wú)毒蛋白Avr1-CO39，并且RGA5-A也能與Avr1-CO39互作[62]。這些結(jié)果揭示了同一抗病基因能夠識(shí)別多個(gè)不同無(wú)毒基因的新機(jī)制，至于形成這種識(shí)別機(jī)制的原因和進(jìn)化動(dòng)力是什么，目前尚不清楚。

圖1 水稻稻瘟病抗性基因與相應(yīng)無(wú)毒基因互作模式

2.3 R-Avr基因非直接互作介導(dǎo)的ETI激活模式

AvrPiz-t是廣譜抗稻瘟病基因Piz-t對(duì)應(yīng)的無(wú)毒基因。研究表明，AvrPiz-t與Piz-t無(wú)直接相互作用。AvrPiz-t編碼一個(gè) N端含信號(hào)肽、長(zhǎng)度為 108個(gè)氨基酸的新分泌蛋白[58]。生物學(xué)功能研究發(fā)現(xiàn)，AvrPiz-t具有毒性，能夠抑制 BAX誘導(dǎo)的煙草(N. benthamiana)細(xì)胞死亡。水稻中異源表達(dá)AvrPiz-t則增強(qiáng)了水稻對(duì)稻瘟病的感病性，并顯著抑制flg22和幾丁質(zhì)誘導(dǎo)的PTI反應(yīng)[68]。此外，利用酵母雙雜交技術(shù)，以AvrPiz-t為誘餌，篩選水稻cDNA文庫(kù)，鑒定了 12個(gè) AvrPiz-t互作蛋白，命名為 APIPs(Avr-Piz-t-interacting proteins)；對(duì)其中RING finger型泛素連接酶基因APIP6的功能進(jìn)行研究，發(fā)現(xiàn)AvrPiz-t通過(guò)干擾APIP6的泛素連接酶活性而抑制APIP6介導(dǎo)的 PTI反應(yīng)，反之 APIP6能夠泛素化 AvrPiz-t；生物學(xué)功能研究表明，APIP6RNAi水稻增強(qiáng)了對(duì)稻瘟菌的感病性[68]。這些結(jié)果揭示了稻瘟菌效應(yīng)蛋白通過(guò)干擾植物泛素蛋白降解系統(tǒng)而抑制植物抗病性的新機(jī)制。

3 抗稻瘟病育種新策略

3.1 全基因組選擇育種

隨著新一代高通量測(cè)序技術(shù)的發(fā)展，開(kāi)展大規(guī)模水稻重測(cè)序、發(fā)掘覆蓋全基因組的高密度SNP標(biāo)記已成為可能。最近，全基因組關(guān)聯(lián)分析(Genome wide association studies，GWAS)技術(shù)在水稻、玉米(Zea mays)等作物基因定位中已成功應(yīng)用[69～72]。例如，Huang等[73]利用二代測(cè)序技術(shù)對(duì)517份我國(guó)水稻種質(zhì)材料進(jìn)行重測(cè)序，利用識(shí)別的全基因組 SNP標(biāo)記對(duì)14個(gè)性狀進(jìn)行了 GWAS分析，定位了與這些性狀相關(guān)的 80個(gè)基因位點(diǎn)。最近，Huang等[69]又對(duì)100份我國(guó)粳稻品種和來(lái)自33個(gè)國(guó)家的330份水稻材料進(jìn)行了重測(cè)序，結(jié)合之前 517份材料的數(shù)據(jù)，對(duì)共 947份材料的抽穗期和產(chǎn)量性狀進(jìn)行了GWAS分析，發(fā)現(xiàn)了32個(gè)新的與抽穗、產(chǎn)量性狀相關(guān)位點(diǎn)。此外，Zhao等[74]利用基于水稻重測(cè)序開(kāi)發(fā)的44M的SNP芯片對(duì)413份來(lái)自82個(gè)國(guó)家的水稻材料的34個(gè)性狀進(jìn)行 GWAS分析，鑒定了大量水稻性狀相關(guān)基因。這些研究極大地推動(dòng)了以全基因組高密度分子標(biāo)記和大規(guī)模群體表型數(shù)據(jù)為基礎(chǔ)的全基因組選擇(Genome-wide selection，GWS)育種技術(shù)的應(yīng)用[75,76]。在稻瘟病抗性基因GWAS研究中，Zhao等[74]也鑒定了5個(gè)抗性基因位點(diǎn)。最近，Kang等[77]利用來(lái)自 5個(gè)國(guó)家的 5個(gè)稻瘟菌小種對(duì) Zhao等[74]研究中413份水稻材料進(jìn)行了GWAS分析，鑒定了66個(gè)稻瘟病抗性位點(diǎn)，其中53個(gè)為新鑒定的位點(diǎn)。這些結(jié)果表明，利用GWAS 進(jìn)行水稻抗病分子育種具備可行性。

3.2 靶基因調(diào)控技術(shù)育種應(yīng)用

3.2.1 TALEN和CRISPR技術(shù)

TALEN(Transcription activator-like effector nucleases)是新近發(fā)展的利用 TAL效應(yīng)子識(shí)別靶基因特定序列，并結(jié)合核酸內(nèi)切酶對(duì)靶DNA進(jìn)行切割的一種基因編輯技術(shù)[78,79]。目前已在人類細(xì)胞、酵母(Saccharomyces)、斑馬魚(yú)(Danio rerio)、大鼠(Rattusnorvegicus)、小鼠(Musculus)、擬南芥和水稻等生物中成功應(yīng)用。最近，Li等[80]運(yùn)用TALEN技術(shù)準(zhǔn)確敲除了水稻白葉枯病感病基因Os11N3，成功獲得了抗白葉枯病水稻。與TALEN技術(shù)繁瑣的載體構(gòu)建和組裝過(guò)程相比，最新發(fā)展的 CRISPR(Clustered regularly interspaced short palindromic repeats /CRISPR –associated 9，CRISPR/Cas9)技術(shù)則彌補(bǔ)了這一缺點(diǎn)[81]。它是一種RNA介導(dǎo)的基因組定點(diǎn)編輯技術(shù)，具有構(gòu)建簡(jiǎn)單、基因編輯準(zhǔn)確率高的特點(diǎn)[82～84]。該技術(shù)也已在人類細(xì)胞、小鼠、斑馬魚(yú)、果蠅(Drosophila)、線蟲(chóng)(Nematoda)、擬南芥及水稻等多個(gè)物種中成功應(yīng)用。TALEN和CRISPR技術(shù)的應(yīng)用將極大地推動(dòng)水稻基因靶向改良分子育種的發(fā)展。

3.2.2 宿主誘導(dǎo)的基因沉默技術(shù)

宿主誘導(dǎo)的基因沉默(Host Induced Gene Silence，HIGS)技術(shù)是新近發(fā)現(xiàn)的一種利用RNAi策略抑制病原菌侵染的技術(shù)。該技術(shù)是將病原菌致病相關(guān)基因的RNAi沉默片段異源表達(dá)于寄主植物體內(nèi)，通過(guò)誘導(dǎo)入侵病原菌中靶基因的 RNAi沉默，而抑制病原菌的侵染的[85,86]。目前已在大麥、小麥抗白粉病等研究中獲得成功[85,86]。最近，Wang等[87]在水稻中利用 HIGS技術(shù)對(duì)稻瘟菌相關(guān)致病基因進(jìn)行了初步研究，并獲得了 HIGS抗病性增強(qiáng)的轉(zhuǎn)基因水稻，表明HIGS技術(shù)可以用于水稻抗病育種。

3.2.3 病原菌誘導(dǎo)的基因表達(dá)調(diào)控(PIGR)技術(shù)

很多抗病相關(guān)基因過(guò)表達(dá)和 RNAi沉默后，除了增強(qiáng)抗病性外，還引起植株矮化、不育或細(xì)胞死亡等生長(zhǎng)發(fā)育連鎖負(fù)效應(yīng)，使得轉(zhuǎn)基因植物失去育種利用價(jià)值。近來(lái)，研究者發(fā)現(xiàn)一些受病原菌誘導(dǎo)表達(dá)的啟動(dòng)子，如在接種稻瘟菌12 h后顯著誘導(dǎo)表達(dá)的OsQ16水稻基因啟動(dòng)子[88]。我們可以利用這類啟動(dòng)子獲得轉(zhuǎn)基因植物，正常條件下，靶基因不表達(dá)或者低水平表達(dá)，植株正常生長(zhǎng)發(fā)育。當(dāng)病原菌侵染時(shí)，靶基因被瞬時(shí)過(guò)表達(dá)或 RNAi沉默，并迅速激活抗病信號(hào)，以抑制病菌侵染，而后表達(dá)恢復(fù)正常，不影響正常生長(zhǎng)發(fā)育。這種病原菌誘導(dǎo)的基因表達(dá)調(diào)控(Pathogen induced gene regulation，PIGR)技術(shù)，即病原菌誘導(dǎo)的基因過(guò)表達(dá)(Pathogen induced gene overexpression，PIGO)和病原菌誘導(dǎo)的基因沉默(Pathogen induced gene silencing，PIGS)，將在植物抗病育種中具有重要應(yīng)用前景。

4 展 望

近年來(lái)，人們?cè)谡J(rèn)識(shí)水稻抗稻瘟病分子機(jī)制方面取得了一系列重要的進(jìn)展，特別是克隆和鑒定了一批PTI和ETI信號(hào)調(diào)控的關(guān)鍵基因。此外，GWAS、TALEN、CRISPR、HIGS等技術(shù)的發(fā)展，為水稻抗病分子育種提供了新的重要方法，但也帶來(lái)了一些新的問(wèn)題和挑戰(zhàn)。

(1) 水稻抗稻瘟病持久抗性分子機(jī)制是什么？盡管NBS-LRR基因Pb1和Pi35的克隆，在認(rèn)識(shí)水稻持久抗性機(jī)制方面取得了重要的突破[47,54]。但對(duì)于持久抗稻瘟病分子機(jī)制的認(rèn)識(shí)仍有限。此外，Pi35是已克隆主效基因Pish的復(fù)等位基因，這為 Wang等[89]揭示的“水稻持久抗病性需要主效基因和微效基因的共同參與”提供了更為直接的證據(jù)。但持久抗病調(diào)控中主效基因與微效基因介導(dǎo)的信號(hào)途徑是否具有共性？還有待深入探討。

(2) R蛋白與Avr蛋白互作后激活的下游信號(hào)途徑是什么？盡管目前已克隆了22個(gè)R基因和10個(gè)Avr基因。但是，R蛋白與相應(yīng)的Avr蛋白識(shí)別后激活下游信號(hào)途徑是什么？R蛋白，特別是NBS-LRR蛋白，直接調(diào)控的下游靶標(biāo)是什么？仍知之甚少。最近研究發(fā)現(xiàn)，NBS-LRR蛋白 Pb1與轉(zhuǎn)錄因子OsWRKY45直接互作，而激活防衛(wèi)反應(yīng)[90]，為揭示R蛋白調(diào)控的信號(hào)途徑提供了新的重要證據(jù)。但要系統(tǒng)認(rèn)識(shí)水稻R蛋白介導(dǎo)的抗病信號(hào)途徑，有待深入研究。

(3)Avr基因在水稻中的靶調(diào)控基因是什么，它是怎樣調(diào)控這些靶基因介導(dǎo)的抗病信號(hào)的？盡管已有少數(shù)研究表明，稻瘟菌Avr基因能夠干擾水稻抗病信號(hào)，如AvrPiz-t通過(guò)干擾E3泛素連接酶APIP6，而抑制水稻PTI反應(yīng)[68]。但其他Avr基因是否也具有類似功能？其在水稻中的靶標(biāo)是什么？是怎樣調(diào)控水稻抗病信號(hào)的？都有待系統(tǒng)研究。

(4)Avr基因檢測(cè)技術(shù)能否應(yīng)用到水稻抗病品種選育及布局上？目前，已定位了超過(guò) 40個(gè)稻瘟菌Avr基因[91]，其中10個(gè)已克隆。因此，是否可以利用分子檢測(cè)技術(shù)適時(shí)檢測(cè)田間稻瘟菌群Avr基因頻率和變化動(dòng)態(tài)，為地區(qū)性抗病品種選育、布局提供科學(xué)指導(dǎo)？將成為一項(xiàng)值得開(kāi)發(fā)利用的技術(shù)。

(5) 如何將新的分子育種技術(shù)與常規(guī)育種技術(shù)有效結(jié)合，以提高抗病品種選擇效率，加速育種進(jìn)程？盡管新發(fā)展的GWAS、TALEN、CRISPR、HIGS等為開(kāi)發(fā)新的分子育種技術(shù)提供了重要信息。但如何將它們與常規(guī)育種程序科學(xué)有序結(jié)合，發(fā)展一套系統(tǒng)、高效、低成本而便于育種家利用的技術(shù)體系，仍是一個(gè)難題。

[1]Dean R, Van Kan JA, Pretorius ZA, Hammond-Kosack KE,Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R,Ellis J, Foster GD. The Top 10 fungal pathogens in molecular plant pathology.Mol Plant Pathol, 2012, 13(4):414–430.

[2]Skamnioti P, Gurr SJ. Against the grain: safeguarding rice from rice blast disease.Trends Biotechnol, 2009, 27(3):141–150.

[3]Jones JDG, Dangl JL. The plant immune system.Nature,2006, 444(7117): 323–329.

[4]Boller T, He SY. Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens.Science, 2009,324(5928): 742–744.

[5]Akerley BJ, Cotter PA, Miller JF. Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction.Cell, 1995, 80(4): 611–620.

[6]Kunze G, Zipfel C, Robatzek S, Niehaus K, Boller T, Felix G. The N terminus of bacterial elongation factor Tu elicits innate immunity inArabidopsisplants.Plant Cell, 2004,16(12): 3496–3507.

[7]Dow M, Newman MA, von Roepenack E. The induction and modulation of plant defense responses by bacterial lipopolysaccharides.Annu Rev Phytopathol, 2000, 38:241–261.

[8]Erbs G, Silipo A, Aslam S, De Castro C, Liparoti V, Flagiello A, Pucci P, Lanzetta R, Parrilli M, Molinaro A. Peptidoglycan and muropeptides from pathogens agrobacterium and xanthomonas elicit plant innate immunity:structure and activity.Chem Biol, 2008, 15(5): 438–448.

[9]Asai T, Tena G, Plotnikova J, Willmann MR, Chiu W-L,Gomez-Gomez L, Boller T, Ausubel FM, Sheen J. MAP kinase signalling cascade inArabidopsisinnate immunity.Nature, 2002, 415(6875): 977–983.

[10]Qi Y, Tsuda K, Glazebrook J, Katagiri F. Physical association of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) immune receptors in Arabidopsis.Mol Plant Pathol, 2011, 12(7): 702–708.

[11]Thomma BPHJ, Nürnberger T, Joosten MHAJ. Of PAMPs and effectors: the blurred PTI-ETI dichotomy.Plant Cell,2011, 23(1): 4–15.

[12]Felix G, Duran JD, Volko S, Boller T. Plants have a sensitive perception system for the most conserved domain of bacterial flagellin.Plant J, 1999, 18(3): 265–276.

[13]Takai R, Isogai A, Takayama S, Che FS. Analysis of flagellin perception mediated by flg22 receptor OsFLS2 in rice.Mol Plant Microbe Interact, 2008, 21(12): 1635–1642.

[14]Sun Y, Li L, Macho AP, Han Z, Hu Z, Zipfel C, Zhou JM,Chai J. Structural basis for flg22-induced activation of the Arabidopsis FLS2-BAK1 immune complex.Science, 2013,342(6158): 624–628.

[15]Lu D, Wu S, Gao X, Zhang Y, Shan L, He P. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity.Proc Natl Acad Sci USA, 2010, 107(1): 496–501.

[16]Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S,Ntoukakis V, Jones JD, Shirasu K, Menke F, Jones A. Direct regulation of the NADPH oxidase RBOHD by the PRR-Associated kinase BIK1 during plant immunity.Mol Cell, 2014, 54(1): 43–55.

[17]Li L, Li M, Yu LP, Zhou ZY, Liang XX, Liu ZX, Cai GH,Gao LY, Zhang XJ, Wang YC, Chen S, Zhou JM. The FLS2-Associated kinase BIK1 directly phosphorylates the NADPH oxidase RbohD to control plant immunity.Cell Host Microbe, 2014, 15(3): 329–338.

[18]Li D, Wang L, Wang M, Xu YY, Luo W, Liu YJ, Xu ZH,Li J, Chong K. Engineering OsBAK1 gene as a molecular tool to improve rice architecture for high yield.Plant Biotechnol J, 2009, 7(8): 791–806.

[19]張慧娟. 磷酸-1-鞘氨醇在植物抗病反應(yīng)中的作用及水稻和擬南芥 BIK1 在逆境反應(yīng)中的功能分析[學(xué)位論文]. 杭州: 浙江大學(xué), 2009.

[20]Miya A, Albert P, Shinya T, Desaki Y, Ichimura K, Shirasu K, Narusaka Y, Kawakami N, Kaku H, Shibuya N. CERK1,a LysM receptor kinase, is essential for chitin elicitor signaling inArabidopsis.Proc Natl Acad Sci USA, 2007,104(49): 19613–19618.

[21]Iizasa Ei, Mitsutomi M, Nagano Y. Direct binding of a plant LysM receptor-like kinase, LysM RLK1/CERK1,to chitin in vitro.J Biol Chem, 2010, 285(5): 2996–3004.

[22]Petutschnig EK, Jones AM, Serazetdinova L, Lipka U,Lipka V. The lysin motif receptor-like kinase (LysM-RLK)CERK1 is a major chitin-binding protein in Arabidopsis thaliana and subject to chitin-induced phosphorylation.J Biol Chem, 2010, 285(37): 28902–28911.

[23]Kaku H, Nishizawa Y, Ishii-Minami N, Akimoto-Tomiyama C, Dohmae N, Takio K, Minami E, Shibuya N.Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor.Proc Natl Acad Sci USA, 2006, 103(29): 11086–11091.

[24]Shimizu T, Nakano T, Takamizawa D, Desaki Y, Ishii-Minami N, Nishizawa Y, Minami E, Okada K, Yamane H,Kaku H, Shibuya N. Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice.Plant J, 2010, 64(2): 204–214.

[25]Akamatsu A, Wong HL, Fujiwara M, Okuda J, Nishide K,Uno K, Imai K, Umemura K, Kawasaki T, Kawano Y,Shimamoto K. An OsCEBiP/OsCERK1-OsRacGEF1-OsRac1 module is an essential early component of chitin-induced rice immunity.Cell Host Microbe, 2013, 13(4): 465–476.

[26]Kawano Y, Akamatsu A, Hayashi K, Housen Y, Okuda J,Yao A, Nakashima A, Takahashi H, Yoshida H, Wong HL,Kawasaki T, Shimamoto K. Activation of a Rac GTPase by the NLR family disease resistance protein Pit plays a critical role in rice innate immunity.Cell Host Microbe, 2010, 7(5): 362–375.

[27]Kawano Y, Shimamoto K. Early signaling network in rice PRR-mediated and R-mediated immunity.Curr Opin Plant Biol, 2013, 16(4): 496–504.

[28][28]Liu B, Li JF, Ao Y, Qu J, Li Z, Su J, Zhang Y, Liu J,Feng D, Qi KB, He YM, Wang JF, Wang HB. Lysin motif-containing proteins LYP4 and LYP6 play dual roles in peptidoglycan and chitin perception in rice innate immunity.Plant Cell, 2012, 24(8): 3406–3419.

[29]Bateman A, Bycroft M. The structure of a LysM domain from E. coli membrane-bound lytic murein transglycosylase D (MltD).J Mol Biol, 2000, 299(4): 1113–1119.

[30]Silipo A, Erbs G, Shinya T, Dow JM, Parrilli M, Lanzetta R, Shibuya N, Newman MA, Molinaro A. Glyco-conjugates as elicitors or suppressors of plant innate immunity.Glycobiology, 2010, 20(4): 406–419.

[31]Willmann R, Lajunen HM, Erbs G, Newman MA, Kolb D,Tsuda K, Katagiri F, Fliegmann J, Bono JJ, Cullimore JV,Jehle AK, G?tz F, Kulik A, Molinaro A, Lipka V, Gust AA,Nürnberger T.Arabidopsislysin-motif proteins LYM1 LYM3 CERK1 mediate bacterial peptidoglycan sensing and immunity to bacterial infection.Proc Natl Acad Sci USA, 2011, 108(49): 19824–19829.

[32]Shinya T, Motoyama N, Ikeda A, Wada M, Kamiya K,Hayafune M, Kaku H, Shibuya N. Functional characterization of CEBiP and CERK1 homologs inArabidopsisand rice reveals the presence of different chitin receptor systems in plants.Plant Cell Physiol, 2012, 53(10): 1696–1706.

[33]Faulkner C, Petutschnig E, Benitez-Alfonso Y, Beck M,Robatzek S, Lipka V, Maule AJ. LYM2-dependent chitin perception limits molecular flux via plasmodesmata.Proc Natl Acad Sci USA, 2013, 110(22): 9166–9170.

[34]Chen XW, Shang JJ, Chen DX, Lei CL, Zou Y, Zhai WX,Liu GZ, Xu JC, Ling ZZ, Cao G, Ma BT, Wang YP, Zhao XF, Li SG, Zhu LH. AB ‐ lectin re ceptor kinase gene conferring rice blast resistance.Plant J, 2006, 46(5): 794–804.

[35]Fukuoka S, Saka N, Koga H, Ono K, Shimizu T, Ebana K,Hayashi N, Takahashi A, Hirochika H, Okuno K, Yano M.Loss of function of a proline-containing protein confers durable disease resistance in rice.Science, 2009, 325(5943):998–1001.

[36]Wang ZX, Yano M, Yamanouchi U, Iwamoto M, Monna L,Hayasaka H, Katayose Y, Sasaki T. The Pib gene for rice blast resistance belongs to the nucleotide binding and leucine-rich repeat class of plant disease resistance genes.Plant J, 1999, 19(1): 55–64.

[37]Bryan GT, Wu K-S, Farrall L, Jia Y, Hershey HP, McA-dams SA, Faulk KN, Donaldson GK, Tarchini R, Valent B.A single amino acid difference distinguishes resistant and susceptible alleles of the rice blast resistance gene Pi-ta.Plant Cell, 2000, 12(11): 2033–2046.

[38]Qu SH, Liu GF, Zhou B, Bellizzi M, Zeng LR, Dai LY,Han B, Wang GL. The broad-spectrum blast resistance gene Pi9 encodes a nucleotide-binding site–leucine-rich repeat protein and is a member of a multigene family in rice.Genetics, 2006, 172(3): 1901–1914.

[39]Zhou B, Qu SH, Liu GF, Dolan M, Sakai H, Lu GD, Bellizzi M, Wang GL. The eight amino-acid differences within three leucine-rich repeats between Pi2 and Piz-t resistance proteins determine the resistance specificity toMagnaporthe grisea.Mol Plant Microbe Interact, 2006,19(11): 1216–1228.

[40]Liu X, Lin F, Wang L, Pan Q. The in silico map-based cloning of Pi36, a rice coiled-coil-nucleotide-binding site- eucine-rich repeat gene that confers race-specific resistance to the blast fungus.Genetics, 2007, 176(4): 2541– 2549.

[41]Lin F, Chen S, Que Z, Wang L, Liu X, Pan Q. The blast resistance gene Pi37 encodes a nucleotide binding siteleucine-rich repeat protein and is a member of a resistance gene cluster on rice chromosome 1.Genetics, 2007,177(3): 1871–1880.

[42]Ashikawa I, Hayashi N, Yamane H, Kanamori H, Wu J,Matsumoto T, Ono K, Yano M. Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance.Genetics, 2008, 180(4): 2267–2276.

[43]Hayashi K, Yoshida H. Refunctionalization of the ancient rice blast disease resistance genePitby the recruitment of a retrotransposon as a promoter.Plant J, 2009, 57(3):413–425.

[44]Lee SK, Song MY, Seo YS, Kim HK, Ko S, Cao PJ, Suh JP,Yi G, Roh JH, Lee S,An G, Hahn TR, Wang GL, Ronald P,Jeon JS. Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil–nucleotide-binding–leucine-rich repeat genes.Genetics, 2009,181(4): 1627–1638.

[45]Shang J, Tao Y, Chen X, Zou Y, Lei C, Wang J, Li X, Zhao X, Zhang M, Lu Z, Xu J, Cheng Z, Wan J, Zhu L. Identification of a new rice blast resistance gene, Pid3, by genomewide comparison of paired nucleotide-binding site–leucine-rich repeat genes and their pseudogene alleles between the two sequenced rice genomes.Genetics, 2009, 182(4):1303–1311.

[46]Takahashi A, Hayashi N, Miyao A, Hirochika H. Unique features of the rice blast resistance Pish locus revealed by large scale retrotransposon-tagging.BMC Plant Biol, 2010,10(1): 175.

[47]Hayashi N, Inoue H, Kato T, Funao T, Shirota M, Shimizu T, Kanamori H, Yamane H, Hayano-Saito Y, Matsumoto T,Yano M, Takatsuji H. Durable panicle blast‐ resistance gene Pb1 encodes an atypical CC-NBS-LRR protein and was generated by acquiring a promoter through local genome duplication.Plant J, 2010, 64(3): 498–510.

[48]Rai AK, Kumar SP, Gupta SK, Gautam N, Singh NK,Sharma TR. Functional complementation of rice blast resistance gene Pi-k h (Pi54) conferring resistance to diverse strains ofMagnaporthe oryzae.J Plant Biochem Biotech,2011, 20(1): 55–65.

[49]Zhai C, Lin F, Dong ZQ, He XY, Yuan B, Zeng XS, Wang L, Pan QH. The isolation and characterization of Pik, a rice blast resistance gene which emerged after rice domestication.New Phytol, 2011, 189(1): 321–334.

[50]Yuan B, Zhai C, Wang WJ, Zeng XS, Xu XK, Hu HQ, Lin F, Wang L, Pan QH. The Pik-p resistance to Magnaporthe oryzae in rice is mediated by a pair of closely linked CC-NBS-LRR genes.Theor Appl Genet, 2011, 122(5):1017–1028.

[51]Okuyama Y, Kanzaki H, Abe A, Yoshida K, Tamiru M,Saitoh H, Fujibe T, Matsumura H, Shenton M, Galam DC,Undan J, Ito A, Sone T, Terauchi R. A multifaceted genomics approach allows the isolation of the rice Pia ‐blast resistance gene consisting of two adjacent NBS-LRR protein genes.Plant J, 2011, 66(3): 467–479.

[52]Chen J, Shi YF, Liu WZ, Chai RY, Fu YP, Zhuang JY, Wu JL. A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae.J Genet Genomics, 2011,38(5): 209–216.

[53]Hua L, Wu JZ, Chen CX, Wu WH, He XY, Lin F, Wang L,Ashikawa I, Matsumoto T, Wang L, Pan QH. The isolation of Pi1, an allele at the Pik locus which confers broad spectrum resistance to rice blast.Theor Appl Genet, 2012,125(5): 1047–1055.

[54]Fukuoka S, Yamamoto SI, Mizobuchi R, Yamanouchi U,Ono K, Kitazawa N, Yasuda N, Fujita Y, Nguyen TTT,Koizumi S, Sugimoto K, Matsumoto T, Yano M. Multiple functional polymorphisms in a single disease resistance gene in rice enhance durable resistance to blast.Scientific Reports, 2014, 4,doi:10.1038/srep04550.

[55]Kang S, Sweigard JA, Valent B. The PWL host specificity gene family in the blast fungus Magnaporthe grisea.Mol Plant Microbe Interact, 1995, 8(6): 939–948.

[56]Sweigard JA, Carroll AM, Kang S, Farrall L, Chumley FG,Valent B. Identification, cloning, and characterization of PWL2, a gene for host species specificity in the rice blast fungus.Plant Cell, 1995, 7(8): 1221–1233.

[57]Orbach MJ, Farrall L, Sweigard JA, Chumley FG, Valent B.A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta.Plant Cell, 2000, 12(11):2019–2032.

[58]Li W, Wang BH, Wu J, Lu GD, Hu YJ, Zhang X, Zhang ZG, Zhao Q, Feng Q, Zhang HY, Wang ZY, Wang GL,Han B, Wang ZH,Zhou B. The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t.Mol Plant Microbe Interact, 2009, 22(4):411–420.

[59]Miki S, Matsui K, Kito H, Otsuka K, Ashizawa T, Yasuda N, Fukiya S, Sato J, Hirayae K, Fujita Y, Nakajima T,Tomita F,Sone T. Molecular cloning and characterization of the AVR ‐Pia locus from a Japanese field isolate of Magnaporthe oryzae.Mol Plant Pathol, 2009, 10(3):361–374.

[60]Yoshida K, Saitoh H, Fujisawa S, Kanzaki H, Matsumura H, Yoshida K, Tosa Y, Chuma I, Takano Y, Win J, Kamoun S, Terauchi R. Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae.Plant Cell, 2009, 21(5): 1573–1591.

[61]Leong SA. The ins and outs of host recognition ofMagnaporthe oryzae// Genomics of Disease Stadler Genetics Symposia Series. New York: Springer, 2008: 199–216.

[62]Cesari S, Thilliez G, Ribot C, Chalvon V, Michel C, Jauneau A, Rivas S, Alaux L, Kanzaki H, Okuyama Y, Morel JB, Fournier E, Tharreau D, Terauchi R, Kroj T. The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1- CO39 by direct binding.Plant Cell, 2013, 25(4): 1463–1481.

[63]B?hnert HU, Fudal I, Dioh W, Tharreau D, Notteghem JL,Lebrun MH. A putative polyketide synthase/peptide synthetase from Magnaporthe grisea signals pathogen attack to resistant rice.Plant Cell, 2004, 16(9): 2499–2513.

[64]Bao JW, Tang M, Zhu X, Wang H, Jeon JS, Han SS, Zhou B. Cloning of AvrPi9 by genome gomparison of a pair of putative wild and mutant strains: an important step toward the understanding of the mechanism underlying the broad-spectrum resistance mediated by the rice blast resistance gene Pi9. International Rice Blast Conference.Jeju, korea2013

[65]Kou YQ, Zhou B, Naqvi NI. A secreted effector confers avirulence towards Pi9-mediated broad-spectrum blast resistance.International Rice Blast Conference. Jeju, Korea2013.

[66]Jia YL, McAdams SA, Bryan GT, Hershey HP, Valent B.Direct interaction of resistance gene and avirulence gene products confers rice blast resistance.EMBO J, 2000,19(15): 4004–4014.

[67]Kanzaki H, Yoshida K, Saitoh H, Fujisaki K, Hirabuchi A,Alaux L, Fournier E, Tharreau D, Terauchi R. Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions.Plant J, 2012, 72(6): 894–907.

[68]Park CH, Chen S, Shirsekar G, Zhou B, Khang CH,Songkumarn P, Afzal AJ, Ning Y, Wang R, Bellizzi M,Valent B, Wang GL. The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 Ubiquitin Ligase APIP6 to suppress pathogen-associated molecular pattern–triggered immunity in rice.Plant Cell, 2012, 24(11): 4748–4762.

[69]Huang XH, Zhao Y, Wei XH, Li CY, Wang AH, Zhao Q, Li WJ, Guo YN, Deng LW, Zhu CR, Fan DL, Lu YQ, Weng QJ, Liu KY, Zhou TY, Jing YF, Si LZ, Dong GJ, Huang T,Lu TT, Feng Q, Qian Q, Li JY, Han B. Genome-wide association study of flowering time and grain yield traits in a worldwide collection of rice germplasm.Nat Genet, 2012,44(1): 32–39.

[70]Huang XH, Lu TT, Han B. Resequencing rice genomes: an emerging new era of rice genomics.Trends Genet, 2013,29(4): 225–232.

[71]Kump KL, Bradbury PJ, Wisser RJ, Buckler ES, Belcher AR, Oropeza-Rosas MA, Zwonitzer JC, Kresovich S,McMullen MD, Ware D, Balint-Kurti PJ, Holland JB.Genome-wide association study of quantitative resistance to southern leaf blight in the maize nested association mapping population.Nat Genet, 2011, 43(2): 163–168.

[72]Tian F, Bradbury PJ, Brown PJ, Hung H, Sun Q,Flint-Garcia S, Rocheford TR, McMullen MD, Holland JB,Buckler ES. Genome-wide association study of leaf architecture in the maize nested association mapping population.Nat Genet, 2011, 43(2): 159–162.

[73]Huang XH, Wei XH, Sang T, Zhao Q, Feng Q, Zhao Y, Li CY, Zhu CR, Lu TT, Zhang ZW, Li M, Fan DL, Guo YL,Wang AH, Wang L, Deng LW, Li WJ, Lu YQ, Weng QJ,Liu KY, Huang Tao, Zhou TY, Jing YF, Li Wei, Lin Zhang,Buckler ES, Qian Q, Zhang QF, Li JY, Han B. Genome-wide association studies of 14 agronomic traits in rice landraces.Nat Genet, 2010, 42(11): 961–967.

[74]Zhao K, Tung CW, Eizenga GC, Wright MH, Ali ML,Price AH, Norton GJ, Islam MR, Reynolds A, Mezey J,McClung AM, Bustamante CD, McCouch SR. Genome-wide association mapping reveals a rich genetic architecture of complex traits inOryza sativa.Nat Commun,2011, 2: 467,doi: 10.1038/ncomms1467.

[75]Meuwissen THE, Hayes BJ, Goddard ME. Prediction of total genetic value using genome-wide dense marker maps.Genetics, 2001, 157(4): 1819–1829.

[76]Daetwyler HD, Villanueva B, Bijma P, Woolliams JA. Inbreeding in genome‐ wide selection.J Anim Breed Genet,2007, 124(6): 369–376.

[77]Kang H, Zhang Y, Wang Y, Xiao Y, Wang D, Bellizzi M,Qu S, Korniliev P, Mezey JG, LiuW, Wang Z, Yan S, Li Z,Leung H, McCouch S, Wang GL. Molecular dissection of the complex genetic architecture of rice immunity to the blast fungusMagnaporthe oryzaeusing genome-wide association study. International Rice Blast Conference.Jeju, Korea2013.

[78]Christian M, Cermak T, Doyle EL, Schmidt C, Zhang F,Hummel A, Bogdanove AJ, Voytas DF. Targeting DNA double-strand breaks with TAL effector nucleases.Genetics, 2010, 186(2): 757–761.

[79]Bogdanove AJ, Voytas DF. TAL effectors: customizable proteins for DNA targeting.Science, 2011, 333(6051):1843–1846.

[80]Li T, Liu B, Spalding MH, Weeks DP, Yang B.High-efficiency TALEN-based gene editing produces disease-resistant rice.Nat Biotechnol, 2012, 30(5): 390–392.

[81]Pennisi E. The CRISPR craze.Science, 2013, 341(6148):833–836.

[82]Gaj T, Gersbach CA, Barbas III CF. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.Trends Biotechnol, 2013, 31(7): 397–405.

[83]Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW,Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell, 2013, 153(4): 910–918.

[84]Wei CX, Liu JY, Yu ZS, Zhang B, Gao GJ, Jiao RJ.TALEN or Cas9–rapid, efficient and specific choices for genome modifications.J Genet Genomics, 2013, 40(6):281–289.

[85]Nowara D, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D, Hensel G, Kumlehn J, Schweizer P. HIGS: hostinduced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis.Plant Cell, 2010, 22(9):3130–3141.

[86]Nunes CC, Dean RA. Host-induced gene silencing: a tool for understanding fungal host interaction and for developing novel disease control strategies.Mol Plant Pathol,2012, 13(5): 519–529.

[87]Wang MY, Wang XL, Wang GL. Development of a hostinduced gene silencing system to engineer resistant transgenic rice to the rice blast fungusMagnaporthe oryzae.International Rice Blast Conference. Jeju, Korea2013.

[88]王光, 吳智丹, 張磊, 劉鳳權(quán), 邵敏. 水稻稻瘟病菌誘導(dǎo)表達(dá)啟動(dòng)子 OsQ16p的克隆與功能分析. 作物學(xué)報(bào),2012, 38(6): 980–987.

[89]Wang GL, Mackill DJ, Bonman JM, McCouch SR, Champoux MC, Nelson RJ. RFLP mapping of genes conferring complete and partial resistance to blast in a durably resistant rice cultivar.Genetics, 1994, 136(4): 1421–1434.

[90]Inoue H, Hayashi N, Matsushita A, Xinqiong L, Nakayama A, Sugano S, Jiang CJ, Takatsuji H. Blast resistance of CC-NB-LRR protein Pb1 is mediated by WRKY45 through protein–protein interaction.Proc Natl Acad Sci USA, 2013, 110(23): 9577–9582.

[91]Zhang S, Xu JR. Effectors and effector delivery inMagnaporthe oryzae.PLoS Pathog, 2014, 10(1): e1003826.