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

土壤杆菌Ti质粒毒力（vir）和T-DNA区域是植物细胞转化中的主要参与者。 在vir基因表达激活后，农杆菌合成T-DNA区域的单链（ss）拷贝，即T链。 然后，该T链穿过几个屏障，细菌的内外膜和壁，植物细胞膜和壁，以及植物核膜。 为了保护和促进它的旅行，T链被提议作为ssDNA-蛋白质复合物（T复合物）旅行。 VirD 2蛋白结合到T-链的5'端，并且非序列特异性ssDNA结合蛋白VirE 2沿T-链的长度沿着包覆T-链以产生未折叠的和薄的结构。 T-复合物的蛋白质组分可能作为氨基酸序列的来源，所述氨基酸序列在T-链转运期间作为特异性亚细胞定位的信号。 为了支持这一点，已经建立了VirD 2和VirE 2蛋白，以包含共有类型的二分核定位信号（NLS）序列，这导致它们的有效核摄取。 该提案有四个目标：1）直接证明由VirD 2和VirE 2介导的单链DNA复合物的核摄取; 2）开发体外测定以监测T-链整合到植物核DNA中; 3）研究VirB膜复合物的结构和功能，所述VirB膜复合物被提议形成从细菌细胞输出T-复合物的通道;以及，4）鉴定参与农杆菌感染的植物细胞组分。这些结果将为理解农杆菌不愿意转化重要单子叶作物的原因提供重要信息，并将确定有效的转化策略。

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.