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

Jasper, Fred; Koncz, Csaba; Schell, Jeff; Steinbiß, Hans‐Henning

doi:10.1073/pnas.91.2.694

Cited by 47 publications

(31 citation statements)

References 35 publications

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“…Cleavage of these doublestranded border sequences requires VirD1 and VirD2 proteins, both in vivo (82,99,155,369) and in vitro (281). In vitro, however, VirD2 protein alone can cleave a single-stranded T-DNA border sequence (154,249). Cleavage of the 25-bp T-DNA border results predominantly from the nicking of the T-DNA "lower strand," as conventionally presented, between nucleotides 3 and 4 of the border sequence (301,353).…”

Section: Molecular Basis Of Agrobacterium-mediated Transformation Whamentioning

confidence: 99%

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

Gelvin

2003

Microbiol Mol Biol Rev

1,169

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: Molecular Basis Of Agrobacterium-mediated Transformation Whamentioning

confidence: 99%

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

Gelvin

2003

Microbiol Mol Biol Rev

1,169

687

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“…In vitro, VirD2 alone nicks ss oligonucleotides bearing the T-DNA border sequence (Jasper et al, 1994;Pansegrau et al, 1993a). In the presence of an excess of cleavage products, VirD2 can also catalyze the reverse reaction, joining two pieces of ssDNA (Pansegrau et al, 1993a).…”

Section: Nic Sites and Nicking Enzymes (Table 1 Item 8)mentioning

confidence: 99%

The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights

et al. 2000

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This review is dedicated to Jeff Schell, one of the founders of modern`Agrobiology', the genetic and molecular dissection of crown gall disease. Together with notable scientists at the University of Gent, Belgium, Jeff spearheaded the discovery of the Ti-plasmid. The elusive`tumor inducing principle' was uncloaked and provided impetus for an incredibly fruitful subsequent 25 years of analyses. Scientists all over the world were caught up in unraveling the underlying mechanisms of Agrobacterium-mediated gene transfer to plants, and along the way uncovered a movable feast of fundamental insights. Below we summarize a sampling of Agrobacterium's most recently recognized accomplishments.

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“…It is not clear yet whether bacterial and/or plant factors mediate the integration of T-DNA. VirD2 has been shown not only to cleave the border sequence of ssDNA in vitro but also to rejoin the reaction partners (15,23). However, although VirD2-mediated in vitro cutting and rejoining reactions are T-DNA border specific, T-DNA integration in vivo is sequence independent (11,18,19,31).…”

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Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

Ziemienowicz

Tinland

Bryant

et al. 2000

Molecular and Cellular Biology

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Agrobacterium tumefaciens, a gram-negative soil bacterium, transfers DNA to many plant species. In the plant cell, the transferred DNA (T-DNA) is integrated into the genome. An in vitro ligation-integration assay has been designed to investigate the mechanism of T-DNA ligation and the factors involved in this process. The VirD2 protein, which is produced in Agrobacterium and is covalently attached to T-DNA, did not, under our assay conditions, ligate T-DNA to a model target sequence in vitro. We tested whether plant extracts could ligate T-DNA to target oligonucleotides in our test system. The in vitro ligation-integration reaction did indeed take place in the presence of plant extracts. This reaction was inhibited by dTTP, indicating involvement of a plant DNA ligase. We found that prokaryotic DNA ligases could substitute for plant extracts in this reaction. Ligation of the VirD2-bound oligonucleotide to the target sequence mediated by T4 DNA ligase was less efficient than ligation of a free oligonucleotide to the target. T-DNA ligation mediated by a plant enzyme(s) or T4 DNA ligase requires ATP.The soil bacterium Agrobacterium tumefaciens is a plant pathogen responsible for tumor induction on dicotyledonous plants through its ability to transfer DNA carrying plant-active oncogenes to the plant cell (16,25,36). Because of this ability, Agrobacterium is widely used for plant transformation (12). The transferred DNA (T-DNA), which in Agrobacterium resides on a large tumor-inducing (Ti) plasmid, is processed within the bacterium and is exported to the plant, where it is integrated into the plant genome (28,29,30). Proteins encoded by the virulence (vir) region of the Ti plasmid regulate T-DNA processing and transfer. Virulence proteins recognize 24-bplong imperfect direct repeats (border sequences) that define the T-DNA. In the presence of the VirD1 protein, VirD2 cleaves the border sequence in a site-and strand-specific manner and concomitantly becomes covalently attached to the 5Ј end of the nicked DNA (9, 13, 33, 35). The nicked DNA is then displaced 5Ј to 3Ј from the plasmid, producing single-stranded T-DNA (8). The T-DNA-VirD2 complex and the VirE2 protein are believed to be transferred to the plant with the help of a pilus-like structure containing the VirB and VirD4 proteins (2, 4, 10, 37). In the plant cell, T-DNA is coated with the single-stranded DNA (ssDNA)-binding protein VirE2 (8, 25), forming a T-DNA-protein complex that is imported into the nucleus, where the T-DNA is integrated into the nuclear genome. The VirD2 protein is transferred into the nucleus in conjunction with the T-DNA; it presumably remains attached to it up to the integration step.In higher eukaryotic organisms, such as plants, illegitimate recombination is the predominant mechanism of integration for naked DNA (20,22,24,26). Likewise, the T-DNA is integrated into the plant genome by illegitimate recombination, a mechanism in which two DNA molecules that do not share extensive homology are joined (11,18,19,31). It is not clear yet whe...

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

Cited by 47 publications

References 35 publications

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

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

The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights

Plant Enzymes but Not Agrobacterium VirD2 Mediate T-DNA Ligation In Vitro

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