Characterization of T-Circles and Their Formation Reveal Similarities to Agrobacterium T-DNA Integration Patterns.
Characterization of T-Circles and Their Formation Reveal Similarities to Agrobacterium T-DNA Integration Patterns.
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DOI：
10.3389/fpls.2022.849930
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发表时间：
2022
影响因子：
5.6
通讯作者：
Gelvin, Stanton B.
中科院分区：
文献类型：
--
作者：
Singer, Kamy;Lee, Lan-Ying;Yuan, Jing;Gelvin, Stanton B.
农杆菌将T-DNA转移到植物中，在那里它可能整合到基因组中。非同源末端连接(NHEJ)被认为是T-DNA整合的机制，但各种NHEJ蛋白的作用仍存在争议。NHEJ在T-DNA整合中的作用的遗传证据产生了相互矛盾的结果。我们建议研究T环的形成，作为理解T-DNA整合的代理。T环是在植物中形成的环状双链T-DNA分子，连接在其左(Lb)和右(Rb)边界区域。我们表征了在烟草或拟南芥中形成的数百个T环的Lb-Rb结合区。这些连接类似于在整合的T-DNA中发现的T-DNA/植物DNA连接：在由多个T-DNA分子组成的复杂T-环中，Rb-Rb/Lb-Lb连接占主导地位；Lb处的缺失比Rb处的缺失更频繁和广泛；连接位点经常使用微同源；边界之间经常存在来自植物基因组或各种农杆菌复制子的填充DNA。KU80不是有效形成T环所必需的，而病毒D2DNA突变对T环形成和T-ω整合的影响也类似。我们认为，研究T环的形成可以作为了解T-DNA整合的替代方法。
Agrobacterium transfers T-DNA to plants where it may integrate into the genome. Non-homologous end-joining (NHEJ) has been invoked as the mechanism of T-DNA integration, but the role of various NHEJ proteins remains controversial. Genetic evidence for the role of NHEJ in T-DNA integration has yielded conflicting results. We propose to investigate the formation of T-circles as a proxy for understanding T-DNA integration. T-circles are circular double-strand T-DNA molecules, joined at their left (LB) and right (RB) border regions, formed in plants. We characterized LB-RB junction regions from hundreds of T-circles formed in Nicotiana benthamiana or Arabidopsis thaliana. These junctions resembled T-DNA/plant DNA junctions found in integrated T-DNA: Among complex T-circles composed of multiple T-DNA molecules, RB-RB/LB-LB junctions predominated over RB-LB junctions; deletions at the LB were more frequent and extensive than those at the RB; microhomology was frequently used at junction sites; and filler DNA, from the plant genome or various Agrobacterium replicons, was often present between the borders. Ku80 was not required for efficient T-circle formation, and a VirD2 ω mutation affected T-circle formation and T-DNA integration similarly. We suggest that investigating the formation of T-circles may serve as a surrogate for understanding T-DNA integration.
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