Gene targeting and instability of <i>Agrobacterium</i> T‐DNA loci in the plant genome

Risseeuw, Eddy; Dijk, Marry E.I. Franke-van; Hooykaas, Paul J. J.

doi:10.1046/j.1365-313x.1997.11040717.x

Cited by 59 publications

(33 citation statements)

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“…However, gene targeting in plants by homologous recombination has been at best extremely inefficient (72,173,223,237,238,269,270). An alternative system for gene targeting is the use of site-specific integration systems such as Cre-lox.…”

Section: T-dna Integration and Transgene Expressionmentioning

confidence: 99%

“…The ability to perform gene replacement experiments has become a staple of bacterial, fungal, and even animal cell and molecular biology research. However, homologous recombination in plants generally occurs at 10 Ϫ5 the frequency of illegitimate recombination (71,173,223,237,238,269,270). We need an Agrobacterium-mediated transformation system that delivers T-DNA to the plant nucleus efficiently, but is deficient in random T-DNA integration.…”

Section: Prospectsmentioning

confidence: 99%

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Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Gelvin

2003

Microbiol Mol Biol Rev

1,167

687

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SUMMARY Agrobacterium tumefaciens and related Agrobacterium species have been known as plant pathogens since the beginning of the 20th century. However, only in the past two decades has the ability of Agrobacterium to transfer DNA to plant cells been harnessed for the purposes of plant genetic engineering. Since the initial reports in the early 1980s using Agrobacterium to generate transgenic plants, scientists have attempted to improve this “natural genetic engineer” for biotechnology purposes. Some of these modifications have resulted in extending the host range of the bacterium to economically important crop species. However, in most instances, major improvements involved alterations in plant tissue culture transformation and regeneration conditions rather than manipulation of bacterial or host genes. Agrobacterium-mediated plant transformation is a highly complex and evolved process involving genetic determinants of both the bacterium and the host plant cell. In this article, I review some of the basic biology concerned with Agrobacterium-mediated genetic transformation. Knowledge of fundamental biological principles embracing both the host and the pathogen have been and will continue to be key to extending the utility of Agrobacterium for genetic engineering purposes.

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Section: T-dna Integration and Transgene Expressionmentioning

confidence: 99%

Section: Prospectsmentioning

confidence: 99%

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Gelvin

2003

Microbiol Mol Biol Rev

1,167

687

View full text Add to dashboard Cite

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“…In most of these studies, DNA was introduced into cultured cells by direct gene transfer (10,11) or Agrobacterium-mediated transformation (12)(13)(14)(15). In general, GT efficiencies were low, on both a per-cell basis (Ͻ10 Ϫ7 GT events per cell) and per-integration basis (10 Ϫ6 to 10 Ϫ4 GT events per integration), and negative selection to select against random integrations was not successful (16)(17)(18)(19). Two exceptional studies reported relatively high GT ratios (Ϸ10 Ϫ3 GT events per integration), but these successes have not been repeated (20,21).…”

Section: ϫ5mentioning

confidence: 99%

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

Lloyd

Plaisier

Carroll³

et al. 2005

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

386

249

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Targeted mutagenesis is an essential tool of reverse genetics that could be used experimentally to investigate basic plant biology or modify crop plants for improvement of important agricultural traits. Although targeted mutagenesis is routine in several model organisms including yeast and mouse, efficient and widely usable methods to generate targeted modifications in plant genes are not currently available. In this study we investigated the efficacy of a targeted-mutagenesis approach based on zinc-finger nucleases (ZFNs). In this procedure, ZFNs are used to generate double-strand breaks at specific genomic sites, and subsequent repair produces mutations at the break site. To determine whether ZFNs can cleave and induce mutations at specific sites within higher plant genomes, we introduced a construct carrying both a ZFN gene, driven by a heat-shock promoter, and its target into the Arabidopsis genome. Induction of ZFN expression by heat shock during seedling development resulted in mutations at the ZFN recognition sequence at frequencies as high as 0.2 mutations per target. Of 106 ZFN-induced mutations characterized, 83 (78%) were simple deletions of 1-52 bp (median of 4 bp), 14 (13%) were simple insertions of 1-4 bp, and 9 (8%) were deletions accompanied by insertions. In 10% of induced individuals, mutants were present in the subsequent generation, thus demonstrating efficient transmission of the ZFNinduced mutations. These data indicate that ZFNs can form the basis of a highly efficient method for targeted mutagenesis of plant genes.gene targeting ͉ nonhomologous end joining A major focus of plant biotechnology is genetic modification and improvement of crop plants. With this aim, large-scale genome and͞or EST sequencing projects are underway for many important plant species including rice, maize, wheat, soybean, and tomato (www.ncbi.nlm.nih.gov͞genomes͞PLANTS͞ PlantList.html). The enormous amount of genome-sequence information becoming available has intensified the need for methods that can use this sequence information to generate targeted modifications in plant genes. Targeted-mutagenesis methods could be used experimentally to investigate plant gene function or for genetic modification of important crop plants. Such methods are especially important for species, including most crops, that lack readily available mutant collections (1, 2). Furthermore, targeted mutagenesis could facilitate development of genetically modified crops lacking transgenic DNA, including genes conferring resistance to antibiotics. To date, efficient and widely usable methods for targeted modification of higher plant genomes are not available.The most widely used targeted-mutagenesis strategy is gene targeting (GT) by homologous recombination (3-6). Efficient GT procedures have been available for Ͼ20 years in yeast (7) and mouse (8). In these systems, DNA fragments are introduced into cultured cells, and repair by homologous recombination causes the introduced DNA to be incorporated into the homologous locus. Typically, GT events ...

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“…This contrasts sharply with the relatively efficient sitespecific integration of bacterial plasmids (Albert et al, 1995). Similarly, gene targeting in plant cells using T-DNA has been demonstrated although at disappointingly low frequencies (Miao and Lam, 1995;Risseeuw et al, 1997;Wang et al 2001). Engineering T-DNA in order to accomplish more precise, non-random transformation still remains elusive.…”

Section: Introductionmentioning

confidence: 99%

“…This process is called agroinfection (Grimsley et al, 1986) and the replicative release of the viral DNA molecules has been postulated as a major recombination pathway (Stenger et al, 1991); however, homologous recombination within T-DNA (Lazarowitz et al, 1989) as well as illegitimate recombination involving the T-DNA border sequences (Bakkeren et al, 1989) has also been implicated in this process. Homologous recombination of T-DNA molecules also occurs during plant gene targeting (Risseeuw et al, 1997;Wang et al, 2001). Thus, it seems that T-DNA molecules are active in a variety of recombination activities.…”

Section: Introductionmentioning

confidence: 99%

T‐DNA recombination and replication in maize cells

Zhao

Coats

et al. 2003

The Plant Journal

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SummaryT-DNA recombination and replication was analyzed in 'black mexican sweet' (BMS) cells transformed with T-DNAs containing the replication system from wheat dwarf virus (WDV). Upon recombination between the T-DNA ends, a promoterless marker gene (gusA) was activated. Activation of the recombination marker gene was delayed and increased exponentially over time, suggesting that recombination and amplification of the T-DNA occurred in maize cells. Mutant versions of the viral initiator gene (rep), known to be defective in the replication function, failed to generate recoverable recombinant T-DNA molecules. Circularization of T-DNA by the FLP/FRT site-specific recombination system and/or homologous recombination was not necessary to recover circular T-DNAs. However, replicating T-DNAs appeared to be suitable substrates for site-specific and homologous recombination. Among 33 T-DNA border junctions sequenced, only one pair of identical junction sites was found implying that the population of circular T-DNAs was highly heterogenous. Since no circular T-DNA molecules were detected in treatments without rep, it suggested that T-DNA recombination was linked to replication and might have been stimulated by this process. The border junctions observed in recombinant T-DNA molecules were indicative of illegitimate recombination and were similar to left-border recombination of T-DNA into the genome after Agro-mediated plant transformation. However, recombination between T-DNA molecules differed from T-DNA/genomic DNA junction sites in that few intact right borders were observed. The replicating T-DNA molecules did not enhance genomic random integration of T-DNA in the experimental configuration used for this study.

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Gene targeting and instability of Agrobacterium T‐DNA loci in the plant genome

Cited by 59 publications

References 0 publications

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

Targeted mutagenesis using zinc-finger nucleases in Arabidopsis

T‐DNA recombination and replication in maize cells

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