Rice blast caused by Magnaporthe oryzae is the most destructive fungal disease in crops, greatly threatening rice production and food security worldwide. The identification and utilization of broad-spectrum resistance genes are considered to be the most economic and effective method to control the disease. In the past decade, many blast resistance (R) genes have been identified, which mainly encode nucleotide-binding leucine-rich repeat (NLR) receptor family and confer limited race-specific resistance to the fungal pathogen. Resistance genes conferring broad-spectrum blast resistance are still largely lacking. In this study, we carried out a map-based cloning of the new blast R locus Pizh in variety ZH11. A bacterial artificial chromosome (BAC) clone of 165 kb spanning the Pizh locus was sequenced and identified 9 NLR genes, among which only Pizh-1 and Pizh-2 were expressed. Genetic complementation experiments indicated that Pizh-1 but not Pizh-2 alone could confer blast resistance. Intriguingly, both mutations on Pizh-1 and Pizh-2 by CRISPR-Cas9 abolished the Pizh-mediated resistance. We also observed that Pizh-1-mediated resistance was partially dependent on Pizh-2. Pizh-1 and Pizh-2 form a complex of NLRs through direct interaction. This suggests that Pizh-1 may function as the executor NLR and Pizh-2 as a ‘helper’ NLR that shares functional redundancy with other NLRs. Our current study provides not only a good tool for rice disease resistance breeding but also deep insight into NLR association and function in plant immunity.
稻瘟病是由稻瘟病菌引起的最具破壞性的真菌病害，嚴(yán)重威脅著世界范圍內(nèi)的水稻生產(chǎn)和糧食安全。廣譜抗性基因的鑒定和利用被認(rèn)為是最經(jīng)濟(jì)、最有效的防治方法。過去的十年里，人們發(fā)現(xiàn)了許多稻瘟病抗性(R)基因，這些基因主要編碼核苷酸結(jié)合的富亮氨酸重復(fù)序列(NLR)受體家族，對(duì)真菌有限的小種有著特異性抗性，廣譜抗稻瘟病的抗性基因很大程度上仍然缺乏。本研究對(duì)粳稻品種中花11(ZH11)的新抗性位點(diǎn)Pizh進(jìn)行了克隆。對(duì)一個(gè)包含Pizh的165 kb細(xì)菌人工染色體(BAC)克隆進(jìn)行測(cè)序，鑒定出9個(gè)NLR基因，其中只有Pizh-1和Pizh-2表達(dá)。遺傳互補(bǔ)實(shí)驗(yàn)表明，只有Pizh-2而不是Pizh-2對(duì)稻瘟病菌有抗性。有趣的是，通過CRISPR-Cas9對(duì)Pizh-1和Pizh-2的突變，都消除了Pizh介導(dǎo)的抗性。我們也觀察到Pizh-1介導(dǎo)的抗性部分依賴于Pizh-2。Pizh-1和Pizh-2通過直接相互作用形成NLRs復(fù)合物。這表明，Pizh-1可能作為“執(zhí)行者”NLR，而Pizh-2可能作為“助手”NLR，與其他NLRs共享功能冗余。本研究不僅為水稻抗病育種提供了良好的工具，而且為深入研究NLR與植物免疫的關(guān)系和功能提供了新的思路。