T-DNA Export From Agrobacterium to Plant Cells: Biochemical,Cell Biological and Genetic Approaches

农杆菌Ti质粒毒力（vir）和T-DNA区是植物细胞转化的主要参与者。激活vir基因表达后，农杆菌合成T-DNA区域的单链拷贝，即t链。然后t链穿过几个屏障，细菌的内外膜和细胞壁，植物的细胞膜和细胞壁，以及植物的核膜。为了保护和促进它的旅行，t链被建议以ssdna -蛋白质复合物（t复合物）的形式旅行。VirD2蛋白结合到t链的5'端，而非序列特异性的ssDNA结合蛋白VirE2沿着t链的长度包裹t链，产生一个展开的薄结构。t复合物的蛋白质成分可能作为氨基酸序列的来源，在t链运输过程中作为特定亚细胞定位的信号。为了支持这一点，已经建立了VirD2和VirE2蛋白含有共识型二部核定位信号（NLS）序列，这导致它们有效的核摄取。该提案有四个目的：1)直接证明由VirD2和VirE2介导的单链DNA复合物的核摄取；2)建立t链与植物核DNA整合的体外检测方法；3)研究VirB膜复合物的结构和功能，提出形成t复合物从细菌细胞输出的通道；4)鉴定参与农杆菌感染的植物细胞成分。研究结果将为理解为什么农杆菌不愿转化重要的单子叶作物植物提供关键信息，并将确定有效的改造策略。

9507782 Zambryski The Agrobacterium Ti plasmid virulence (vir) and T-DNA regions are the major players in plant cell transformation. Following activation of vir gene expression, Agrobacterium synthesizes a single stranded (ss) copy of the T-DNA region, the T-strand. This T-strand then travels through several barriers, the bacterial inner and outer membranes and wall, the plant cell membrane and wall, and the plant nuclear membrane. To protect and facilitate its travels, the T-strand is proposed to travel as a ssDNA-protein complex, the T-complex. VirD2 protein binds to the 5' end of the T-strand, and the non-sequence specific ssDNA binding protein, VirE2, coats the T-strand along, its length to produce an unfolded and thin structure. The protein components of the T-complex likely serve as a source of amino acid sequences that act as signals for specific subcellular localization during T-strand transport. In support of this, VirD2 and VirE2 proteins have been established to contain consensus type bipartite nuclear localization signal (NLS) sequences, that cause their efficient nuclear uptake. The proposal has four aims: 1) to directly demonstrate nuclear uptake of single stranded DNA complexes mediated by VirD2 and VirE2; 2) to develope an in vitro assay to monitor T-strand integration into plant nuclear DNA; 3) to investigate the structure and function of the VirB membrane complex, proposed to form a channel for T-complex export from the bacterial cell; and , 4) to identify plant cell components involved in Agrobacterium infection. The results will provide information critical to understand why Agrobacterium is reluctant to transform the important monocotyledonous crop plants, and will determine effective modified strategies for their transformation.