Gene editing a constitutively active OsRac1 by homologous recombination-based gene targeting induces immune responses in rice
Gene editing a constitutively active OsRac1 by homologous recombination-based gene targeting induces immune responses in rice
复制标题
通过基于同源重组的基因靶向对组成型活性 OsRac1 进行基因编辑可诱导水稻免疫反应
DOI：
10.1093/pcp/pct147
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
Shimamoto K
中科院分区：
文献类型：
--
作者：
Dang TT;Shimatani Z;Kawano Y;Terada R;Shimamoto K
OsRac1是植物小GTPase Rac/Rop家族成员，在水稻免疫中起关键作用。OsRac1的组成活性（CA） G19V突变先前被证明可以诱导活性氧产生、植物抗毒素合成和防御基因激活，从而抵抗稻瘟病感染。为了进一步研究G19V突变在抗病中的作用，我们通过基因靶向引入单碱基替代，并使用cre - loxpsite特异性重组去除选择标记。基因靶向产生的ca - osrac1基因称为ca - gosrac1。G19V突变从靶载体转移到osrac1位点，并稳定地传递给下一代。纯合子ca - gosrac1植株叶片中突变体转录水平远低于野生型植株。相比之下，根、叶鞘和穗中的突变体转录本比叶片中的突变体转录本更丰富。然而，几丁质处理后，与野生型植物相比，细胞培养中防御相关基因espal1和pbz1的表达量更高。此外，在水稻稻瘟病菌亲和种感染突变植株的叶鞘中观察到诱导超敏反应（HR）样细胞死亡。在ca - gosrac1植物中，许多基因先前被证明是由稻瘟病菌和稻黄单胞菌诱导的。在叶鞘中诱导oryzae（Xoo）侵染，但未发生病原菌侵染。这些结果表明，基因靶向将为基因功能研究和作物改良提供有用的突变。
OsRac1 is a member of the plant small GTPase Rac/Rop family and plays a key role in rice immunity. The constitutively active (CA) G19V mutation of OsRac1 was previously shown to induce reactive oxygen species production, phytoalexin synthesis and defense gene activation, leading to resistance to rice blast infection. To study further the effect of the G19V mutation in disease resistance, we introduced a single base substitution by gene targeting and removed the selectable marker using Cre-loxPsite-specific recombination. TheCA-OsRac1gene generated by gene targeting was termedCA-gOsRac1. The G19V mutation was transferred from a targeting vector to theOsRac1locus and stably transmitted to the next generation. In the leaf blade of homozygousCA-gOsRac1plants, mutant transcript levels were much lower than in those of wild-type plants. In contrast, mutant transcripts in roots, leaf sheaths and panicles were more abundant than those in leaf blades. However, upon chitin treatment, the expression of defense-related genesPAL1andPBZ1in the cell culture was greater in the mutants compared with wild-type plants. Furthermore, induction of hypersensitive response (HR)-like cell death was observed in the leaf sheaths of mutant plants infected with a compatible race of rice blast fungus. In theCA-gOsRac1plants, a number of genes previously shown to be induced byMagnaporthe oryzaeandXanthomonas oryzaepv.oryzae(Xoo) infection were induced in the leaf sheath without pathogen infection. These results suggest that gene targeting will provide mutations useful for gene function studies and crop improvement.