Agrobacterium Tumefaciens-Mediated Transformation: Patterns of T-Dna Integration Into the Host Genome

Windels, Pieter; Buck, Sylvie De; Depicker, Anna

doi:10.1007/978-0-387-72290-0_12

Cited by 17 publications

(15 citation statements)

References 153 publications

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“…Transgenic loci obtained after A. tumefaciens transformation are considered as less complex than those obtained after direct gene transfer, but integration of multiple T-DNA copies is fairly common (Tzfira et al, 2004;and references therein;Windels et al, 2008;and references therein). These multiple T-DNA copies are usually integrated into a limited number of loci and are arranged as direct or inverted repeat configurations (Holsters et al, 1983;Jorgensen et al, 1987;Jouanin et al, 1989;De Block and Debrouwer, 1991;De Neve et al, 1997;Krizkova and Hrouda, 1998;De Buck et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

The T‐DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

et al. 2009

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Transgenic loci obtained after Agrobacterium tumefaciens-mediated transformation can be simple, but fairly often they contain multiple T-DNA copies integrated into the plant genome. To understand the origin of complex T-DNA loci, floral-dip and root transformation experiments were carried out in Arabidopsis thaliana with mixtures of A. tumefaciens strains, each harboring one or two different T-DNA vectors. Upon floral-dip transformation, 6-30% of the transformants were co-transformed by multiple T-DNAs originating from different bacteria and 20-36% by different T-DNAs from one strain. However, these co-transformation frequencies were too low to explain the presence of on average 4-6 T-DNA copies in these transformants, suggesting that, upon floral-dip transformation, T-DNA replication frequently occurs before or during integration after the transfer of single T-DNA copies. Upon root transformation, the co-transformation frequencies of T-DNAs originating from different bacteria were similar or slightly higher (between 10 and 60%) than those obtained after floral-dip transformation, whereas the co-transformation frequencies of different T-DNAs from one strain were comparable (24-31%). Root transformants generally harbor only one to three T-DNA copies, and thus co-transformation of different T-DNAs can explain the T-DNA copy number in many transformants, but T-DNA replication is postulated to occur in most multicopy root transformants. In conclusion, the comparable co-transformation frequencies and differences in complexity of the T-DNA loci after floral-dip and root transformations indicate that the T-DNA copy number is highly determined by the transformation-competent target cells

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Section: Introductionmentioning

confidence: 99%

The T‐DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

et al. 2009

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“…Integration of T-DNA delivered to plant cells by A. tumefaciens shows specific patterns; in most cases, the 5# end of T-DNA is preserved because of protection by covalently attached VirD2 (precise integration), whereas the 3# end of the integrated T-DNA is poorly conserved (truncations of 3-100 nucleotides; Tinland et al, 1995;Tinland, 1996;Windels et al, 2008). Analysis of the intactness of the integrated transgene cassette was performed by PCR using 10 sets of primers specific to various regions of the GUS cassette ( Fig.…”

Section: Intactness Of the Transgenementioning

confidence: 99%

“…Standard procedures of plant transformation using Agrobacterium often result in clear integration patterns, such as a low number of copies of integrated transgenes (Windels et al, 2008), whereas most other methods used in plant biotechnology (direct gene transfer, bombardment, etc.) result in the integration of multiple DNA molecules and, as a consequence, multiple copy or multiple loci insertion patterns that may lead to variations in the transgene expression (Latham et al, 2006).…”

Section: Transgene Integration Patternmentioning

confidence: 99%

“…The integration process apparently requires broken DNA, or at least an area of active replication or transcription (Ziemienowicz et al, 2008). Agrobacterium-mediated transformation is an efficient process and typically, in dicotyledonous plants (dicots), predominantly results in integration of the transgenes at a single locus; integrated T-DNA is mostly intact and allows for normal expression of the transgene (Windels et al, 2008).…”

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A Novel Method of Transgene Delivery into Triticale Plants Using the Agrobacterium Transferred DNA-Derived Nano-Complex

et al. 2012

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Genetic transformation of monocotyledonous plants still presents a challenge for plant biologists and biotechnologists because monocots are difficult to transform with Agrobacterium tumefaciens, whereas other transgenesis methods, such as gold particlemediated transformation, result in poor transgene expression because of integration of truncated DNA molecules. We developed a method of transgene delivery into monocots. This method relies on the use of an in vitro-prepared nano-complex consisting of transferred DNA, virulence protein D2, and recombination protein A delivered to triticale microspores with the help of a Tat 2 cell-penetrating peptide. We showed that this approach allowed for single transgene copy integration events and prevented degradation of delivered DNA, thus leading to the integration of intact copies of the transgene into the genome of triticale plants. This resulted in transgene expression in all transgenic plants regenerated from microspores transfected with the full transferred DNA/protein complex. This approach can easily substitute the bombardment technique currently used for monocots and will be highly valuable for plant biology and biotechnology.Transgenesis, or genetic transformation, finds many applications in plant biology, e.g. in cell biology and gene function studies. Importantly, it also allows for the generation of plants with improved agricultural traits significantly faster than any conventional breeding practice. The technology is based on the delivery of genes of interest from a broad range of sources into a plant genome. Two major transformation techniques include Agrobacterium-mediated DNA delivery and

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“…Second, specific mutations at the VirD2 putative integrase motif resulted in small deletions at the 59 end of the T-DNA molecules (Tinland et al, 1995). Therefore, Tinland et al (1995) revisited the SSGR integration model to the microhomology-dependent integration model, suggesting that only short sequences of the T-DNA molecule actually anneal to preintegration genomic sites (Tzfira et al, 2004a;Windels et al, 2008;Ziemienowicz et al, 2008).…”

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Blocking Single-Stranded Transferred DNA Conversion to Double-Stranded Intermediates by Overexpression of Yeast DNA REPLICATION FACTOR A

et al. 2014

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Agrobacterium tumefaciens delivers its single-stranded transferred DNA (T-strand) into the host cell nucleus, where it can be converted into double-stranded molecules. Various studies have revealed that double-stranded transfer DNA (T-DNA) intermediates can serve as substrates by as yet uncharacterized integration machinery. Nevertheless, the possibility that T-strands are themselves substrates for integration cannot be ruled out. We attempted to block the conversion of T-strands into double-stranded intermediates prior to integration in order to further investigate the route taken by T-DNA molecules on their way to integration. Transgenic tobacco (Nicotiana benthamiana) plants that overexpress three yeast (Saccharomyces cerevisiae) protein subunits of DNA REPLICATION FACTOR A (RFA) were produced. In yeast, these subunits (RFA1-RFA3) function as a complex that can bind single-stranded DNA molecules, promoting the repair of genomic double strand breaks. Overexpression of the RFA complex in tobacco resulted in decreased T-DNA expression, as determined by infection with A. tumefaciens cells carrying the b-glucuronidase intron reporter gene. Gene expression was not blocked when the reporter gene was delivered by microbombardment. Enhanced green fluorescent protein-assisted localization studies indicated that the three-protein complex was predominantly nuclear, thus indicating its function within the plant cell nucleus, possibly by binding naked T-strands and blocking their conversion into double-stranded intermediates. This notion was further supported by the inhibitory effect of RFA expression on the cell-to-cell movement of Bean dwarf mosaic virus, a single-stranded DNA virus. The observation that RFA complex plants dramatically inhibited the transient expression level of T-DNA and only reduced T-DNA integration by 50% suggests that doublestranded T-DNA intermediates, as well as single-stranded T-DNA, play significant roles in the integration process.

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Agrobacterium Tumefaciens-Mediated Transformation: Patterns of T-Dna Integration Into the Host Genome

Cited by 17 publications

References 153 publications

The T‐DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

The T‐DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell

A Novel Method of Transgene Delivery into Triticale Plants Using the Agrobacterium Transferred DNA-Derived Nano-Complex

Blocking Single-Stranded Transferred DNA Conversion to Double-Stranded Intermediates by Overexpression of Yeast DNA REPLICATION FACTOR A

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