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.
