Li Xiaoyang, Zhu Tingting, Tu Haitao, Pan Shen Q
Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
School of Stomatology and Medicine, Foshan Institute of Molecular Bio-Engineering, Foshan University, Foshan, China.
Front Plant Sci. 2020 Apr 21;11:464. doi: 10.3389/fpls.2020.00464. eCollection 2020.
is the causal agent of crown gall disease in nature; in the laboratory the bacterium is widely used for plant genetic modification. The bacterium delivers a single-stranded transferred DNA (T-DNA) and a group of crucial virulence proteins into host cells. A putative T-complex is formed inside host cells that is composed of T-DNA and virulence proteins VirD2 and VirE2, which protect the foreign DNA from degradation and guide its way into the host nucleus. However, little is known about how the T-complex is assembled inside host cells. We combined the split-GFP and split-sfCherry labeling systems to study the interaction of -delivered VirE2 and VirE3 in host cells. Our results indicated that VirE2 co-localized with VirE3 on the cytoplasmic side of the host cellular membrane upon the delivery. We identified and characterized two tandem domains at the VirE3 C-terminus that interacted with VirE2 . Deletion of these two domains abolished the VirE2 accumulation on the host plasma membrane and affected the transformation. Furthermore, the two VirE2-interacting domains of VirE3 exhibited different affinities with VirE2. Collectively, this study demonstrates that the anchorage protein VirE3 uses the two tandem VirE2-interacting domains to facilitate VirE2 protection for T-DNA at the cytoplasmic side of the host cell entrance.
在自然环境中是冠瘿病的致病因子；在实验室中，这种细菌被广泛用于植物基因改造。该细菌将单链转移DNA（T-DNA）和一组关键的毒力蛋白传递到宿主细胞中。在宿主细胞内形成了一个假定的T复合体，它由T-DNA以及毒力蛋白VirD2和VirE2组成，这些蛋白可保护外源DNA不被降解，并引导其进入宿主细胞核。然而，关于T复合体如何在宿主细胞内组装却知之甚少。我们结合了分裂绿色荧光蛋白（split-GFP）和分裂超折叠樱桃荧光蛋白（split-sfCherry）标记系统，以研究细菌传递的VirE2和VirE3在宿主细胞中的相互作用。我们的结果表明，在传递后，VirE2与VirE3在宿主细胞膜的细胞质一侧共定位。我们鉴定并表征了VirE3 C末端的两个串联结构域，它们与VirE2相互作用。删除这两个结构域消除了VirE2在宿主质膜上的积累，并影响了转化。此外，VirE3的两个与VirE2相互作用的结构域与VirE2表现出不同的亲和力。总的来说，这项研究表明，锚定蛋白VirE3利用两个串联的与VirE2相互作用的结构域，在宿主细胞入口的细胞质一侧促进VirE2对T-DNA的保护。
