Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5′ virD gene products.

Abstract

Agrobacterium tumefaciens transfers its Ti‐plasmid T‐DNA to plant cells. This process is initiated by plant‐induced activation of the Ti‐plasmid virulence loci, resulting in the generation of single stranded (ss) cleavages of the Ti‐plasmid T‐DNA border sequences (border nicks) and ss linear unipolar T‐DNA molecules (T‐strands). A single T‐strand is produced from the two‐border T‐region of the pGV3850 nopaline plasmid. In this paper the induced molecular events for the complex T‐region of the pTiA6 octopine plasmid are analyzed. This T‐region carries four T‐DNA borders delimiting three T‐DNA elements (TR, TC and TL). Induction of pTiA6 generates cleavages independently at its border repeats, and six distinct T‐strand species corresponding to TR, TR/TC, TR/TC/TL, TC, TC/TL and TL. These T‐strand molecules are linear and correspond to the bottom strand of the pTiA6 T‐region. Thus, borders can function for both initiation and termination of T‐strand synthesis. We propose that the different pTiA6 T‐strands are independently generated, and that the distribution of border nicks within the parental T‐region determines which T‐strand is produced. To identify genes involved in T‐strand production, pTiA6 virulence (vir) and chromosomal virulence (chv) mutant strains were analyzed. VirA and VirG, the vir regulatory loci are required. Furthermore, the two 5′ cistrons of virD are required for both border nicks and T‐strands, suggesting that these genes encode the border endonuclease, and that T‐strand production is dependent on border nicks. That no mutants are defective for T‐strands alone suggests that functions encoded outside of vir and chv might mediate some of the later reactions of T‐strand synthesis.

The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights

“…In vivo in Agrobacterium, VirD1/D2 are both required for the nicking reaction (Scheiffele et al, 1995;Stachel et al, 1987;Yanofsky et al, 1986). In vitro, VirD2 alone nicks ss oligonucleotides bearing the T-DNA border sequence (Jasper et al, 1994;Pansegrau et al, 1993a).…”

Agrobacterium T-strand production in vitro: sequence-specific cleavage and 5' protection of single-stranded DNA templates by purified VirD2 protein.

“…Studies of the interaction of T-DNA borders with VirDl and VirD2 proteins in either Agrobacterium or Escherichia coli revealed that VirDl is required for efficient binding of VirD2 to the border repeats. The VirDlVirD2 complex is known as a sequence-specific endonuclease-topoisomerase that introduces a nick in the conjugative T-strand (the "bottom strand") at the third base of the 25-bp border (8)(9)(10)(11)(12).…”

Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes

“…A regulatory system consisting of VirA (a transmembrane sensor) and VirG (a transcriptional activator) regulate vir gene expression (24,25,44). Following vir induction, the VirDl and VirD2 proteins provide topoisomerase (19) and endonuclease (42,56) functions that initiate T-DNA processing by introducing a site-and strand-specific nick within conserved 25-bp cis-acting sequences, termed borders, that delineate the ends of the T-DNA molecule (2,31,47). A strand displacement synthesis reaction probably initiates at the 5' border cleavage site to generate a linear, single-stranded molecule (the T strand) corresponding to the bottom strand of the T-DNA between the borders (2,41,42).…”