![]() Together, VirD1 (a helicase) and VirD2 (an endonuclease) bind to and nick DNA at 25-bp directly repeated T-DNA border repeat sequences ( Jayaswal et al., 1987 Wang et al., 1987). (2) Processing T-DNA from the parental Ti- or Ri-plasmid ( virD1 and virD2). In the presence of these sugars, vir genes are more fully induced at lower phenolic concentrations ( Peng et al., 1998). These sugars are perceived by a protein, ChvE, encoded by a gene on the Agrobacterium chromosome. In addition to induction of the vir genes by phenolics, many sugars serve as co-inducers. Activated VirG binds to the vir box sequences preceding each vir gene operon, allowing increased expression of each of these operons ( Pazour and Das, 1990). Activation of VirA by these phenolic inducers initiates a phospho-relay, ultimately resulting in phosphorylation and activation of the VirG protein ( Winans, 1991). Because wounding is important for efficient plant transformation, Agrobacterium can sense a wounded potential host by perceiving these phenolic compounds. VirA and VirG compose a two-component system that responds to particular phenolic compounds produced by wounded plant cells ( Stachel et al., 1986). (1) Sensing plant phenolic compounds and transducing this signal to induce expression of vir genes ( virA and virG). The vir region consists of approximately 10 operons (depending upon the Ti- or Ri-plasmid) that serve four major functions. Several recent reviews enumerate factors involved in and influencing Agrobacterium-mediated transformation ( Gelvin, 2003 McCullen and Binns, 2006). However, some chromosomal genes important for virulence likely mediate the bacterial response to the environment ( Xu and Pan, 2000 Saenkham et al., 2007). Douglas et al., 1985 Cangelosi et al., 1987, 1989 Robertson et al., 1988 Matthysse, 1995 O'Connell and Handelsman, 1999). These chromosomal genes generally are involved in bacterial exopolysaccharide synthesis, maturation, and secretion (e.g. Transfer requires three major elements: (1) T-DNA border repeat sequences (25 bp) that flank the T-DNA in direct orientation and delineate the region that will be processed from the Ti/Ri-plasmid ( Yadav et al., 1982) (2) vir genes located on the Ti/Ri-plasmid and (3) various genes (chromosomal virulence and other genes) located on the bacterial chromosomes. (2000b).Īgrobacterium transfers T-DNA, which makes up a small (approximately 5%–10%) region of a resident Ti-plasmid or root-inducing plasmid (Ri-plasmid), to numerous species of plants ( DeCleene and DeLey, 1976 Anderson and Moore, 1979), although the bacterium can be manipulated in the laboratory to transfer T-DNA to fungal ( Bundock et al., 1995 Piers et al., 1996 de Groot et al., 1998 Abuodeh et al., 2000 Kelly and Kado, 2002 Li et al., 2007) and even animal cells ( Kunik et al., 2001 Bulgakov et al., 2006). Some of these considerations were previously described in a review by Hellens et al. In this review, we recount the history of development of T-DNA binary vector systems, and we describe important components of these systems. This binary system permitted facile manipulation of Agrobacterium and opened up the field of plant genetic engineering to numerous laboratories. However, scientists eventually learned that T-DNA transfer could still be effected if the T-DNA region and the virulence ( vir) genes required for T-DNA processing and transfer were split into two replicons. Initial technologies to introduce genes of interest (goi) into Agrobacterium involved complex microbial genetic methodologies that inserted these goi into the transfer DNA (T-DNA) region of large tumor-inducing plasmids (Ti-plasmids). For more than two decades, scientists have used Agrobacterium-mediated genetic transformation to generate transgenic plants.
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