Department of Molecular, Cellular and Developmental Biology, Faculty of Arts and Sciences, Yale University, New Haven, CT, USA.
Department of Genetics, Yale School of Medicine, Yale University, New Haven, CT, USA.
Nat Plants. 2023 Sep;9(9):1398-1408. doi: 10.1038/s41477-023-01495-w. Epub 2023 Aug 31.
Transformation via Agrobacterium tumefaciens is the predominant method used to introduce exogenous DNA into plant genomes. Transfer DNA (T-DNA) originating from Agrobacterium can be integrated as a single copy or in complex concatenated forms, but the mechanisms affecting final T-DNA structure remain unknown. Here we demonstrate that inclusion of retrotransposon (RT)-derived sequences in T-DNA can increase T-DNA copy number by more than 50-fold in Arabidopsis thaliana. These additional T-DNA copies are organized into large concatemers, an effect primarily induced by the long terminal repeats (LTRs) of RTs that can be replicated using non-LTR DNA repeats. We found that T-DNA concatenation is dependent on the activity of the DNA repair proteins MRE11, RAD17 and ATR. Finally, we show that T-DNA concatenation can be used to increase the frequency of targeted mutagenesis and gene targeting. Overall, this work uncovers molecular determinants that modulate T-DNA copy number in Arabidopsis and demonstrates the utility of inducing T-DNA concatenation for plant gene editing.
农杆菌介导的转化是将外源 DNA 导入植物基因组的主要方法。源自农杆菌的转移 DNA（T-DNA）可以作为单拷贝或复杂串联形式整合，但影响最终 T-DNA 结构的机制仍不清楚。在这里，我们证明了 T-DNA 中包含逆转座子（RT）衍生序列可以使拟南芥中的 T-DNA 拷贝数增加 50 多倍。这些额外的 T-DNA 拷贝被组织成大的串联体，这主要是由 RT 的长末端重复（LTR）诱导的，LTR 可以使用非 LTR DNA 重复进行复制。我们发现 T-DNA 串联依赖于 DNA 修复蛋白 MRE11、RAD17 和 ATR 的活性。最后，我们表明 T-DNA 串联可用于提高靶向诱变和基因靶向的频率。总的来说，这项工作揭示了调节拟南芥 T-DNA 拷贝数的分子决定因素，并证明了诱导 T-DNA 串联用于植物基因编辑的实用性。
