Plant Biology Division, The Samuel Roberts Noble Foundation, Ardmore, Oklahoma 73401, USA.
Plant Cell. 2012 Oct;24(10):4110-23. doi: 10.1105/tpc.112.100495. Epub 2012 Oct 12.
Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)-mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-ray cross complementation group4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.
根癌农杆菌是一种土壤病原体，它通过将其部分肿瘤诱导质粒（T-DNA）转移到植物基因组中，导致许多双子叶植物患上冠瘿病。已经鉴定出几种在农杆菌附着植物细胞和 T-DNA 转运到细胞核中起作用的植物因子，但转化过程中的 T-DNA 整合步骤理解甚少，据推测是通过非同源末端连接（NHEJ）介导的双链 DNA 断裂（DSB）修复发生的。在这里，我们报告了 X 射线交叉互补组 4（XRCC4）的负作用，XRCC4 是 NHEJ 所需的关键蛋白之一，它在农杆菌 T-DNA 整合中起作用。拟南芥和本氏烟中 XRCC4 的下调增加了 T-DNA 的整合，从而增加了稳定转化。拟南芥中 XRCC4 的过表达导致 T-DNA 整合减少，从而导致稳定转化减少。有趣的是，XRCC4 在酵母双杂交系统和体内直接与农杆菌蛋白 VirE2 相互作用。表达 VirE2 的拟南芥植物对 DNA 损伤化学试剂博莱霉素更敏感，并且表现出更高的稳定转化。我们假设 VirE2 滴定或排除了用于 DSB 修复的活性 XRCC4 蛋白，从而延迟了染色体中 DSB 的闭合，为 T-DNA 整合提供了更多的机会。
