水稻抗稻瘟病天然免疫機(jī)制及抗病育種新策略

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，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，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通過磷酸化NADPH氧化酶RbohD，而誘導(dǎo)ROS的產(chǎn)生[16,17]。OsBAK1過表達(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毒基因的克隆

克隆抗稻瘟病基因與其對(duì)應(yīng)的無毒基因，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 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)的無毒基因。研究表明，AvrPiz-t與Piz-t無直接相互作用。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)序開發(fā)的44M的SNP芯片對(duì)413份來自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(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、果蠅(Drosophila)、線蟲(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，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，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，但也帶來了一些新的問題和挑戰(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 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。

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