Agrobacterium tumefaciens-mediated transformation of yeast.

Abstract

Agrobacterium tumefaciens transfers a piece of its Ti plasmid DNA (transferred DNA or T-DNA) into plant cells during crown gall tumorigenesis. A. tumefaciens can transfer its T-DNA to a wide variety of hosts, including both dicotyledonous and monocotyledonous plants. We show that the host range of A. tumefaciens can be extended to include Saccharomyces cerevisiae. Additionally, we demonstrate that while T-DNA transfer into S. cerevisiae is very similar to T-DNA transfer into plants, the requirements are not entirely conserved. The Ti plasmid-encoded vir genes ofA. tumefaciens that are required for T-DNA transfer into plants are also required for T-DNA transfer into S. cerevisiae, as is vir gene induction. However, mutations in the chromosomal virulence genes of A. tumefaciens involved in attachment to plant cells have no effect on the efficiency of T-DNA transfer into S. cerevisiae. We also demonstrate that transformation efficiency is improved 500-fold by the addition of yeast telomeric sequences within the T-DNA sequence.Agrobacterium tumefaciens causes crown gall tumors in plants by transferring a segment of DNA (transferred DNA or T-DNA) from its tumor-inducing (Ti) plasmid to the nucleus of plant cells. The T-DNA becomes integrated into the plant nuclear genome where it functions to give rise to the characteristic tumor (reviewed in refs. 1 and 2). This process depends on the induction of a set of Ti plasmid-encoded virulence (vir) genes. vir genes are induced via the virA/virG two-component regulatory system which senses monosaccharides and phenolic compounds from wounded plants (reviewed in ref.3). The T-DNA is a single-stranded DNA molecule produced by a virDl/D2-encoded site-specific endonuclease that nicks within two 24-bp direct repeat sequences on the Ti plasmid (4). These repeats, termed border sequences, flank the T-DNA. Following cleavage and excision, the T-DNA is coated by the singlestranded DNA binding protein VirE2 (5), and the resulting T-DNA complex is transferred to the plant cell.The mechanism by which the T-DNA complex is transported through the inner and outer bacterial membranes and into the plant cell is not well understood. It is believed on the basis of several lines of evidence that the VirB proteins and VirD4 are involved in T-DNA transport (reviewed in ref. 6). Once the T-DNA complex enters the plant cell, it is targeted to the nucleus via nuclear localization sequences in the VirD2 and VirE2 proteins (7,8). Upon entering the nucleus, the T-DNA is integrated into the plant genome by illegitimate recombination, a process likely mediated by host factors (9).The study of host factors involved in T-DNA transfer has been difficult and would be greatly facilitated by the availability of a host model amenable to genetic manipulation. Given the similarities between T-DNA transfer and conjugative transfer of broad-host-range plasmids (reviewed in refs. 1, 6, and 10), we set out to determine if A. tumefaciens can transfer T-DNA to the yeast Saccharomyces cerevisiae. It has been demo...

Spatial location and requirements for the assembly of the Agrobacterium tumefaciens type IV secretion apparatus

“…The T4S system of the phytopathogen Agrobacterium tumefaciens transfers oncogenic DNA (T-DNA) and proteins to plant cells, causing neoplastic crown gall tumor disease. The A. tumefaciens T4S system can also direct conjugative transfer of an IncQ plasmid between bacteria and T-DNA to yeast and other eukaryotes (3)(4)(5). In addition to DNA, the system delivers at least three proteins (VirE2, VirE3, and VirF) to target cells (6,7).…”

Protein Interactions Involved in Nuclear Import of the Agrobacterium VirE2 Protein in Vivo and in Vitro

“…Moreover, although plants represent the natural hosts for Agrobacterium, this microorganism can also transform a wide range of other eukaryotic species, ranging from fungi (2,3) to human cells (4). This genetic transformation is achieved by transporting a single-stranded copy (T-strand) 1 of the bacterially transferred DNA (T-DNA) from the tumor-inducing plasmid into the plant cell nucleus and then by integration into the host genome (5,6).…”

Suppression of mutant phenotypes of the Agrobacterium tumefaciens VirB11 ATPase by overproduction of VirB proteins

“…This list includes DNA apparently of any length as long as it contains a recognition site for processing by the VirD2 endonuclease (34), the non-self-transmissible IncQ plasmid RSF1010, most likely also as a single-stranded DNA-protein particle (4, 9, 52), the VirE2 single-stranded DNA-binding protein (7,14,35), and VirF, which functions as an important virulence factor for infection of certain plant species (40). Second, recent studies have shown that the T-complex transporter delivers DNA to many dicotyledonous and monocotyledonous plant species as well as to agrobacteria (3,25) and yeast (10,11,36) recipient cells.…”