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Distinct mechanisms for genomic attachment of the 5′ and 3′ ends of Agrobacterium T-DNA in plants
Nature Plants ( IF 18.0 ) Pub Date : 2022-05-09 , DOI: 10.1038/s41477-022-01147-5
Lejon E M Kralemann 1 , Sylvia de Pater 1 , Hexi Shen 2 , Susan L Kloet 3 , Robin van Schendel 3 , Paul J J Hooykaas 1 , Marcel Tijsterman 1, 3
Affiliation  

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant’s nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5′ end. Different from capture of the 3′ end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5′ attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5′ end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5′ end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3′ capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant’s own DNA repair machineries to transform it.



中文翻译:

植物中农杆菌 T-DNA 5' 和 3' 端基因组附着的不同机制

根癌农杆菌是一种能够通过水平基因转移转化植物的致病菌,是当今植物基因工程的首选载体。转移的载体是 T 链,这是一种由细菌蛋白 VirD2 结合的单链 DNA 分子,它将 T-DNA 引导到植物的细胞核中并在那里整合。VirD2 如何从 T-DNA 中移除,以及哪种机制将释放的末端附加到植物基因组上,目前尚不清楚。在这里,使用新开发的技术在拟南芥的体细胞组织中产生数百个 T-DNA 整合, 我们发现了 T-DNA 5' 端基因组捕获的两个冗余机制。不同于捕获 T-DNA 的 3' 端,这是聚合酶 theta 介导的末端连接 (TMEJ) 的专有作用,5' 连接是通过 TMEJ 或规范的非同源末端连接 (cNHEJ) 完成的。我们进一步发现 TMEJ 需要 MRE11,而 cNHEJ 需要 TDP2 来去除 5' 末端阻断蛋白 VirD2。因此,T-DNA 整合在 MRE11 和 TDP2(或其他 cNHEJ 因子)缺陷的植物中严重受损。为了支持 MRE11 和 cNHEJ 特异性作用于 5' 端,我们通过使用能够在 3' 捕获时形成端粒的 T-DNA 证明了双缺陷植物的整合缺陷的挽救。我们的研究为农杆菌如何运作提供了一个机制模型利用植物自身的 DNA 修复机制对其进行改造。

更新日期:2022-05-10
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