Illegitimate recombination in plants: a model for T-DNA integration.

Gheysen, Godelieve; Villarroel, Raimundo; Montagu, Marc Van

doi:10.1101/gad.5.2.287

Cited by 266 publications

(226 citation statements)

References 50 publications

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“…In the four cell lines (A, C, D, and E) for which the junctions were determined, the integrations lie in unrelated sequences of chromosomes 22, 4, 2, and 7, respectively, and thus no preference for specific integration sites could be distinguished. Taken together, our results show that the Mob protein can protect the 5′ end of the transferred DNA and suggest a mechanism of integration similar to what has been reported for the VirD2-bound T-DNA of A. tumefaciens (23,24).…”

Section: B Henselaesupporting

confidence: 62%

Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

Schröder

Schuelein²,

Québatte³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial type IV secretion systems (T4SS) mediate interbacterial conjugative DNA transfer and transkingdom protein transfer into eukaryotic host cells in bacterial pathogenesis. The sole bacterium known to naturally transfer DNA into eukaryotic host cells via a T4SS is the plant pathogen Agrobacterium tumefaciens . Here we demonstrate T4SS-mediated DNA transfer from a human bacterial pathogen into human cells. We show that the zoonotic pathogen Bartonella henselae can transfer a cryptic plasmid occurring in the bartonellae into the human endothelial cell line EA.hy926 via its T4SS VirB/VirD4. DNA transfer into EA.hy926 cells was demonstrated by using a reporter derivative of this Bartonella -specific mobilizable plasmid generated by insertion of a eukaryotic e gfp -expression cassette. Fusion of the C-terminal secretion signal of the endogenous VirB/VirD4 protein substrate BepD with the plasmid-encoded DNA-transport protein Mob resulted in a 100-fold increased DNA transfer rate. Expression of the delivered e gfp gene in EA.hy926 cells required cell division, suggesting that nuclear envelope breakdown may facilitate passive entry of the transferred ssDNA into the nucleus as prerequisite for complementary strand synthesis and transcription of the e gfp gene. Addition of an eukaryotic neomycin phosphotransferase expression cassette to the reporter plasmid facilitated selection of stable transgenic EA.hy926 cell lines that display chromosomal integration of the transferred plasmid DNA. Our data suggest that T4SS-dependent DNA transfer into host cells may occur naturally during human infection with Bartonella and that these chronically infecting pathogens have potential for the engineering of in vivo gene-delivery vectors with applications in DNA vaccination and therapeutic gene therapy.

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Section: B Henselaesupporting

confidence: 62%

Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

Schröder

Schuelein²,

Québatte³

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…AGR via SDSA might provide a general mechanism to explain previously observed data of DNA rearrangements in plants. Filler DNA in spontaneous deletions (62), complex rearrangements flanking T-DNA insertions (17), and deletions, insertions, and inversions observed following repair of ionizing radiation-induced lesions (56) or nonhomologous end-joining of linear DNA (17a) could all be explained by an error-prone repair mechanism such as, or similar to, SDSA. Furthermore, the maize R-r complex locus was proposed to have evolved through transposon-induced rearrangements similar to those described here, i.e., deletions, insertions, and reshuffling of DNA segments (61).…”

Section: Discussionmentioning

confidence: 99%

Abortive Gap Repair: Underlying Mechanism for Ds element Formation

Rubin

Levy

1997

Molecular and Cellular Biology

114

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The mechanism by which the maize autonomous Ac transposable element gives rise to nonautonomous Ds elements is largely unknown. Sequence analysis of native maize Ds elements indicates a complex chimeric structure formed through deletions of Ac sequences with or without insertions of Ac-unrelated sequence blocks. These blocks are often flanked by short stretches of reshuffled and duplicated Ac sequences. To better understand the mechanism leading to Ds formation, we designed an assay for detecting alterations in Ac using transgenic tobacco plants carrying a single copy of Ac. We found frequent de novo alterations in Ac which were excision rather than sequence dependent, occurring within Ac but not within an almost identical Ds element and not within a stable transposase-producing gene. The de novo DNA rearrangements consisted of internal deletions with breakpoints usually occurring at short repeats and, in some cases, of duplication of Ac sequences or insertion of Ac-unrelated fragments. The ancient maize Ds elements and the young Ds elements in transgenic tobacco showed similar rearrangements, suggesting that Ac-Ds elements evolve rapidly, more so than stable genes, through deletions, duplications, and reshuffling of their own sequences and through capturing of unrelated sequences. The data presented here suggest that abortive Ac-induced gap repair, through the synthesis-dependent strand-annealing pathway, is the underlying mechanism for Ds element formation.Dissociation, or Ds, is the first discovered transposable element (TE). It was identified as a maize locus on chromosome 9, where breaks occur in the presence of Activator (Ac), a second gene found at a separate locus (33,34). Subsequent studies showed that Ac can transpose autonomously whereas Ds moves only in the presence of Ac (35,36). In addition, Ac activity can turn into a Ds type of instability, while no occurrences were found of Ds turning into Ac (37, 38). On the basis of these observations, McClintock proposed that Ds nonautonomous elements are derived from Ac through mutations (39). The proposal that Ac and Ds are phylogenetically related has been supported by molecular analysis, as described below, but the mechanism responsible for the conversion of Ac into Ds is still unknown.Ac is a 4.6-kb-long element flanked by 11-bp terminal inverted repeats (TIRs) (12). It encodes an 807-amino-acid protein, the transposase, necessary for both Ac and Ds transposition (28). Ds elements, on the other hand, do not encode a functional transposase but retain regions which are essential for their transposition (6). There are six fully sequenced Ds elements, all of which share with Ac nearly identical TIRs and fall into the following four categories: (i) those with nearly no similarity to Ac, like Ds1 (57); (ii) elements with highly similar subterminal regions but with internal deletions, like Ds9 (46); (iii) double Ds elements where one internally deleted Ds is inserted into another identical Ds in an inverted orientation (9); and (iv) Ds elements that contain bot...

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“…T-DNA integration does not take place by homologous recombination, the most common method of foreign DNA integration in prokaryotes and lower eukaryotes, because no extensive homology between the T-DNA and target sequences has been found. T-DNA therefore integrates by illegitimate recombination (16)(17)(18)(19), the predominant mechanism of DNA integration into the genomes of higher plants (20)(21)(22). However, plant factors involved in illegitimate recombination of T-DNA into the plant genome have not yet been identified.…”

Section: T-dna Transformation ͉ Haplo-insufficiencymentioning

confidence: 99%

An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration

Mysore

Nam

Gelvin

2000

Proc. Natl. Acad. Sci. U.S.A.

155

150

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Agrobacterium tumefaciens genetically transforms plant cells by transferring a portion of the bacterial Ti-plasmid, the T-DNA, to the plant and integrating the T-DNA into the plant genome. Little is known about the T-DNA integration process, and no plant genes involved in integration have yet been identified. We characterized an Arabidopsis mutant generated by T-DNA insertional mutagenesis, rat5, that is resistant to Agrobacterium root transformation. rat5 contains two copies of T-DNA integrated as a tandem direct repeat into the 3 untranslated region of a histone H2A gene, upstream of the polyadenylation signal sequence. Transient and stable ␤-glucuronidase expression data and assessment of the amount of T-DNA integrated into the genomes of wild-type and rat5 Arabidopsis plants indicated that the rat5 mutant is deficient in T-DNA integration. We complemented the rat5 mutation by expressing the RAT5 histone H2A gene in the mutant plant. Overexpression of RAT5 in wild-type plants increased Agrobacterium transformation efficiency. Furthermore, transient expression of a RAT5 gene from the incoming T-DNA was sufficient to complement the rat5 mutant and to increase the transformation efficiency of wild-type Arabidopsis plants.T-DNA transformation ͉ haplo-insufficiency

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Illegitimate recombination in plants: a model for T-DNA integration.

Cited by 266 publications

References 50 publications

Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

Conjugative DNA transfer into human cells by the VirB/VirD4 type IV secretion system of the bacterial pathogen Bartonella henselae

Abortive Gap Repair: Underlying Mechanism for Ds element Formation

An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration

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