Stimulation of Agrobacterium tumefaciens T-DNA transfer by overdrive depends on a flanking sequence but not on helical position with respect to the border repeat

Abstract

T-DNA transfer by Agrobacterium tumefaciens depends on the right border repeat of the T-DNA and is greatly stimulated by overdrive, an adjacent sequence. We report that the function of overdrive does not depend on helical position with respect to the border repeat. A synthetic 24-bp overdrive and a 12-bp region containing a fully conserved 8-bp core overdrive sequence stimulated virulence equally, but full function required additional bases to the left of the 24-bp sequence.Agrobacterium tumefaciens causes crown gall tumors on many dicotyledonous plants when the bacteria infect wounded plant tissue (for reviews, see references 5, 27, and 50). Virulent strains contain a 190-kb tumor-inducing plasmid that carries genes essential for tumorigenesis (40,46). During tumorigenesis, a specific segment of the Ti plasmid, the T-DNA, transfers into plant cells and integrates into plant nuclear DNA (6,7,47). Expression of certain T-DNA genes in plant cells leads to tumorous growth (1,4,16,28,36). Similar 23-bp direct repeat sequences, called border repeats, define the ends of the T-DNA (15,30,48,51). The genes necessary for T-DNA transfer lie in a different region of the Ti plasmid, the virulence (vir) region, and wounded plant cells produce phenolic compounds that induce expression of the vir genes (32,33). Upon induction, the endonuclease specified by the virDI and virD2 genes produces nicks at a specific location in the bottom strand of each border repeat (2,45,49), and linear single-stranded DNA molecules comprising the bottom strand of the T-DNA (T-strands) accumulate in the bacterial cells (2,9,17,34,35). T-DNA transmission requires only a right border repeat in cis, and deletions removing the right border abolish tumorigenesis (13,20,25,29,44). Full virulence also requires a Ti plasmid sequence, overdrive, that lies to the right of this border repeat (24). Deletions that remove overdrive greatly reduce but do not eliminate virulence, and a synthetic 24-bp overdrive sequence significantly increases the activity of a right border repeat (24). Overdrive increases T-strand accumulation (8,9,38,39,41), and the VirCl protein, which also enhances T-strand production, binds to overdrive (37). VirCl protein bound to overdrive may stimulate T-strand production via direct interaction with the VirD endonuclease. Thus, small changes in spacing between the border repeat and overdrive may affect potential interactions between bound VirCl and VirD2 by rotating the proteins onto opposite faces of the DNA helix. We altered the distance between the border repeat and overdrive in approximately half-turn and full-turn increments; these small changes in spacing had little effect on the ability of overdrive to stimulate tumorigenesis.Conservation between overdrive elements flanking the Border-overdrive assay system. To identify sequences required for efficient T-DNA transmission, we developed an assay system to measure the activity of right border-overdrive constructions (24,25). The octopine-type plasmid pTiA6NC contains two adjacent but ...

Plant-Derived Transfer DNAs

“…Although the overdrive element is believed to function in a position-independent manner with respect to the right border (Shurvinton and Ream, 1991), we found that a single basepair insertion between St02 and upstream DNA reduced transformation frequencies of pSIM579 about 2-fold (Fig. 3A).…”

“…We found the enhancing activity of this region to require at least six pyrimidine residues at conserved positions. The sequence and spacing requirements of the ACR domain may explain why certain previously characterized Ti plasmid mutants displayed reduced virulence (Shurvinton and Ream, 1991). For instance, the impaired activity of mutant WR1803 is associated with an insufficient number of pyrimidine residues at conserved positions, and the low efficiency of WR1804 is linked to a lack of adeninerich spacers.…”

Functional domains of Agrobacterium tumefaciens single-stranded DNA-binding protein VirE2

“…These plasmids were introduced into the virE2 null strain WR5000. For quantitative tumorigenesis assays, we infected potato tuber disks (7-mm diameter) as described previously (53). A. tumefaciens was grown overnight at 28ЊC with aeration in YEP broth containing the appropriate antibiotics, harvested by centrifugation, and suspended in phosphatebuffered saline (3 mM monobasic potassium phosphate, 10 mM dibasic sodium phosphate, 120 mM sodium chloride; pH 7.2) (45).…”

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

“…Overdrive sequences enhance the transmission of T-strands to plants, although the molecular mechanism of how this occurs remains unknown (131,156,256,291,336,337,345). Early reports suggested that the VirC1 protein binds to the overdrive sequence and may enhance T-DNA border cleavage by the VirD1/VirD2 endonuclease (322).…”