T-DNA Delivery Research Articles

The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.).

Transfer of T-DNA from Agrobacterium tumefaciens and A. rhizogenes to cells of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) is demonstrated following the inoculation of immature embryos and immature embryo-derived callus. Agrobacterium T-DNA vectors containing the C1/Lc anthocyanin-biosynthesis regulatory genes, the gusA gene or a synthetic green fluorescent protein gene (sgfp-S65T) were constructed from original binary vectors. The visual T-DNA markers were used as cell-autonomous reporters of early Agrobacterium-mediated transformation events in the wheat and barley cells. This localization of the transformed cells revealed a non-random distribution throughout each embryo and callus piece.

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

Virulence proteins VirD1 and VirD2 are subunits of a relaxosome-like protein complex that mediates conjugational transfer of a Ti plasmid segment, the T-DNA, from Agrobacterium into higher plants. The VirD1-VirD2 complex binds to 25-bp repeats at the borders of the T-DNA and catalyzes sequence-specific nicking of the conjugative DNA strand (the T-strand) at the third base of these repeats. Nuclear localization signals present in VirD2 target the T-strand to plant cell nuclei. In addition, VirD2 probably plays a role in the high-frequency integration of the T-DNA into the plant genome by illegitimate recombination. Whereas Agrobacterium transformation of dicots is very efficient, T-DNA integration in most monocots can barely be detected. To develop an artificial T-DNA delivery system for monocots, a technique for efficient in vitro production of T-strand DNAs was established by using VirD1 and VirD2 proteins purified from overexpressing Escherichia coli strains. The topoisomerase-like VirD2 enzyme was shown to mediate precise, sequence-specific cleavage of T-DNA border sequences carried by single-stranded DNA templates, even in the absence of VirD1 protein. During this reaction, VirD2 remains covalently bound to the 5' end of artificial T-strand DNAs. In contrast, VirD2, alone or in complex with VirD1, fails to nick linear double-stranded DNA templates in vitro.

Delivery of T-DNA from the Agrobacterium tumefaciens chromosome into plant cells

The intact T-region of the B6Ti plasmid of Agrobacterium tumefaciens was stepwise cloned into a site in transposon Tn3. In this way a suitable vehicle (Tn1882) was obtained for translocating the T-region to different replicons, i.e., to other plasmids or the chromosome. The IncP plasmid R772::Tn1882 conferred tumorigenicity on Agrobacterium if the virulence genes were provided in trans in the same cell. This result showed that the T-region present on Tn1882 was transferred efficiently to plant cells. Normal tumor development also occurred if the T-region was placed in the chromosome of A. tumefaciens and an R' plasmid was present carrying virA-E or virA-F. We conclude that the plasmid location of the T-region is not a prerequisite for transfer to the plant cell. The apparently normal delivery of the T-DNA from a bacterial chromosomal location supports a model involving a processing step within Agrobacterium effecting transfer of the T-region as a separate entity.