Department of Human Genetics, Leiden University Medical Centre, 2300 RC Leiden, The Netherlands.
Department of Molecular and Developmental Genetics, Institute of Biology, Leiden University, 2333 BE Leiden, The Netherlands.
Nat Plants. 2016 Oct 31;2(11):16164. doi: 10.1038/nplants.2016.164.
Agrobacterium tumefaciens is a pathogenic bacterium, which transforms plants by transferring a discrete segment of its DNA, the T-DNA, to plant cells. The T-DNA then integrates into the plant genome. T-DNA biotechnology is widely exploited in the genetic engineering of model plants and crops. However, the molecular mechanism underlying T-DNA integration remains unknown. Here we demonstrate that in Arabidopsis thaliana T-DNA integration critically depends on polymerase theta (Pol θ). We find that TEBICHI/POLQ mutant plants (which have mutated Pol θ), although susceptible to Agrobacterium infection, are resistant to T-DNA integration. Characterization of >10,000 T-DNA-plant genome junctions reveals a distinct signature of Pol θ action and also indicates that 3' end capture at genomic breaks is the prevalent mechanism of T-DNA integration. The primer-template switching ability of Pol θ can explain the molecular patchwork known as filler DNA that is frequently observed at sites of integration. T-DNA integration signatures in other plant species closely resemble those of Arabidopsis, suggesting that Pol-θ-mediated integration is evolutionarily conserved. Thus, Pol θ provides the mechanism for T-DNA random integration into the plant genome, demonstrating a potential to disrupt random integration so as to improve the quality and biosafety of plant transgenesis.
根癌农杆菌是一种病原细菌，它通过将其 DNA 的一个离散片段 T-DNA 转移到植物细胞中，从而使植物发生转化。然后，T-DNA 整合到植物基因组中。T-DNA 生物技术广泛应用于模式植物和作物的遗传工程中。然而，T-DNA 整合的分子机制尚不清楚。在这里，我们证明了在拟南芥中，T-DNA 整合严重依赖于聚合酶θ（Pol θ）。我们发现，TEBICHI/POLQ 突变体植物（其 Pol θ 发生突变）尽管易受农杆菌感染，但对 T-DNA 整合具有抗性。对超过 10000 个 T-DNA-植物基因组接头的特征分析揭示了 Pol θ 作用的独特特征，也表明基因组断裂处 3'端捕获是 T-DNA 整合的主要机制。Pol θ 的引物-模板切换能力可以解释在整合位点经常观察到的填充 DNA 的分子嵌合体。其他植物物种中的 T-DNA 整合特征与拟南芥非常相似，表明 Pol-θ 介导的整合在进化上是保守的。因此，Pol θ 为 T-DNA 随机整合到植物基因组提供了机制，表明有可能破坏随机整合，从而提高植物转基因的质量和生物安全性。
