The Oryza sativa (rice) resistance gene Pia confers resistance to the blast fungus Magnaporthe oryzae carrying the AVR-Pia avirulence gene. To clone Pia, we employed a multifaceted genomics approach. First, we selected 12 R-gene analog (RGA) genes encoding nucleotide binding site-leucine rich repeats (NBS-LRRs) proteins from a region on chromosome 11 that shows linkage to Pia. By using seven rice accessions, we examined the association between Pia phenotypes and DNA polymorphisms in the 10 genes, which revealed three genes (Os11gRGA3–Os11gRGA5) exhibiting a perfect association with the Pia phenotypes. We also screened ethyl methane sulfonate (EMS)-treated mutant lines of the rice cultivar ‘Sasanishiki’ harboring Pia, and isolated two mutants that lost the Pia phenotype. DNA sequencing of Os11gRGA3–Os11gRGA5 from the two mutant lines identified independent mutations of major effects in Os11gRGA4. The wild-type ‘Sasanishiki’ allele of Os11gRGA4 (SasRGA4) complemented Pia function in both mutants, suggesting that SasRGA4 is necessary for Pia function. However, when the rice cultivar ‘Himenomochi’ lacking Pia was transfected with SasRGA4, the Pia phenotype was not recovered. An additional complementation study revealed that the two NBS-LRR-type R genes, SasRGA4 and SasRGA5, that are located next to each other and oriented in the opposite direction are necessary for Pia function. A population genetics analysis of SasRGA4 and SasRGA5 suggests that the two genes are under long-term balancing selection.
水稻抗病基因Pia對攜帶有無毒基因AVR-Pia的稻瘟病小種具有抗性。為了克隆Pia作者應(yīng)用了多種基因組學(xué)方法。首先從11染色體選取了與Pia連鎖的12個編碼NBS-LRR蛋白的R基因（RGA）。通過7個水稻品系，對10個基因中DNA多態(tài)性與Pia表型進(jìn)行了關(guān)聯(lián)分析，發(fā)現(xiàn)其中3個基因（Os11gRGA3-Os11gRGA5）與Pia表型完美關(guān)聯(lián)。對攜帶Pia基因的品種Sasanishiki用EMS誘變，發(fā)現(xiàn)2個突變株系喪失了Pia表型。對這兩個突變體Os11gRGA3-Os11gRGA5測序發(fā)現(xiàn)Os11gRGA4分別發(fā)生了獨立的突變。Sasanishiki的Os11gRGA4（SasRGA4）基因能夠互補(bǔ)兩個突變的Pia功能，表明SasRGA4對Pia的功能是必需的。但是將SasRGA4轉(zhuǎn)入缺失Pia的水稻品種Himenomochi卻并不能恢復(fù)Pi表型。另外的互補(bǔ)實驗表明另一個與SasRGA4相鄰、方向相反的基因SasRGA5對于Pia功能也是必需的。群體遺傳分析表明SasRGA4和SasRGA5經(jīng)歷了長期的平衡選擇。
結(jié)果：根據(jù)秈稻粳稻基因組序列選擇了12個與Adh1基因連鎖的Pia候選R基因（RGAs）；兩個秈稻品種攜帶還是缺失Pia的判斷；Os11gRGA3-Os11gRGA5內(nèi)的DNA多態(tài)與Pia表型完美關(guān)聯(lián)；兩個Sasanishiki EMS突變體在SasRGA4基因發(fā)生點突變，導(dǎo)致對攜帶無毒基因的稻瘟病菌抗性喪失；在大量粳稻品系中Oa11gRGA4與Pia表型完美關(guān)聯(lián)；SasRGA4互補(bǔ)了Sasasnishiki突變體的Pia功能；SasRGA4和SasRGA5對AVR-Pia依賴的超敏細(xì)胞死亡是必需的；攜有SasRGA4和SasRGA5的穩(wěn)定轉(zhuǎn)基因株系對攜帶AVR-Pia的稻瘟病菌表現(xiàn)出抗性；Pia基因及其等位基因的DNA序列特征。
討論：兩個基因參與了Pia稻瘟病抗性；R基因的快速克隆。