Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

Rossi, Luca; Höhn, Barbara; Tinland, Bruno

doi:10.1073/pnas.93.1.126

Cited by 166 publications

(118 citation statements)

References 36 publications

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“…These data suggest that T-strand is imported into the nucleus by a protein import pathway via its association with VirE2. Other data have been interpreted to suggest that VirE2 does not provide nuclear-targeting but only protects T-strand from nucleolytic degradation inside the plant cell (Rossi et al, 1996); however, the entire virE2 gene was deleted, simultaneously abolishing both protective and nuclear localizing functions.…”

Section: Plant Nuclear Localization Signals (Table 1 Item 10)mentioning

confidence: 99%

“…At the 3¢ end, integration usually includes most of the left border region, although larger deletions are more common at this end. Deletions of up to 1000 bp at the 3¢ end of the T-strand transferred from a virE2 ± strain suggest that VirE2 plays a signi®cant role in protecting the T-strand from nucleolytic degradation (Rossi et al, 1996). The mechanics of integration remain largely unknown.…”

Section: Integration (Table 1 Item 11)mentioning

confidence: 99%

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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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Section: Plant Nuclear Localization Signals (Table 1 Item 10)mentioning

confidence: 99%

Section: Integration (Table 1 Item 11)mentioning

confidence: 99%

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

et al. 2000

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“…VirD2 acts as a pilot protein as it remains covalently attached to the 5Ј end of the T-strand during transport of the T-DNA into the host cell nucleus (Ward and Barnes, 1988). The ssDNA-binding protein VirE2 binds cooperatively to the T-strand and thereby protects it from degradation in the host cell (Rossi et al, 1996). Both VirD2 and VirE2 contain nuclear localization signals that facilitate transport of the T-complex into the nucleus (Zupan et al, 1996;Citovsky et al, 1997;Ziemienowicz et al, 2001).…”

mentioning

confidence: 99%

Recognition of the Agrobacterium tumefaciens VirE2 Translocation Signal by the VirB/D4 Transport System Does Not Require VirE1

et al. 2003

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Agrobacterium tumefaciens uses a type IV secretion system to deliver a nucleoprotein complex and effector proteins directly into plant cells. The single-stranded DNA-binding protein VirE2, the F-box protein VirF and VirE3 are delivered into host cells via this VirB/D4 encoded translocation system. VirE1 functions as a chaperone of VirE2 by regulating its efficient translation and preventing VirE2-VirE2 aggregation in the bacterial cell. We analyzed whether the VirE1 chaperone is also essential for transport recognition of VirE2 by the VirB/D4 encoded type IV secretion system. In addition, we assayed whether translocation of VirF and VirE3, which also forms part of the virE operon, is affected by the absence of VirE1. We employed the earlier developed CRAFT (Cre recombinase Reporter Assay For Translocation) assay to detect transfer of Cre::Vir fusion proteins from A. tumefaciens into plants, monitored by stable reconstitution of a kanamycin resistance marker, and into yeast, screened by loss of the URA3 gene. We show that the C-terminal 50 amino acids of VirE2 and VirE3 are sufficient to mediate Cre translocation into host cells, confirming earlier indications of a C-terminal transport signal. This transfer was independent of the presence or absence of VirE1. Besides, the translocation efficiency of VirF is not altered in a virE1 mutant. The results unambiguously show that the VirE1 chaperone is not essential for the recognition of the VirE2 transport signal by the transport system and the subsequent translocation across the bacterial envelope into host cells.Agrobacterium tumefaciens causes crown gall disease on a wide range of plants by genetic transformation of host cells with a piece of its oncogenic plasmidborne transfer (T)-DNA (for most recent review, see Gelvin, 2003). The expression of the genes located on the T-DNA, which contain plant transcription and translation signals, results in overproduction of the plant hormones auxin and cytokinin and hence increased cell division and the tumor phenotype. A set of accessory virulence or effector proteins (encoded by the vir region on the tumor inducing [Ti] plasmid) is also transported into the transformed cells to confer full virulence. The host range is not confined to the plant kingdom, because A. tumefaciens can also transform yeast (Bundock et al., 1995), fungi (de Groot et al., 1998), and mammalian cells (Kunik et al., 2000).The translocated proteins of A. tumefaciens described to date include VirD2, VirE2, VirE3, and VirF. In the bacterium, VirD2 introduces a nick at the border sequences surrounding the T-DNA and by replacement synthesis a single-stranded (ss) DNA copy of the bottom strand is released. VirD2 acts as a pilot protein as it remains covalently attached to the 5Ј end of the T-strand during transport of the T-DNA into the host cell nucleus (Ward and Barnes, 1988). The ssDNA-binding protein VirE2 binds cooperatively to the T-strand and thereby protects it from degradation in the host cell (Rossi et al., 1996). Both VirD2 and VirE2 contain nucl...

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“…Error bars indicate SE of two or three biological replicates. Histone H2A-1 Cannot Substitute for VirE2 during Agrobacterium-Mediated Plant Transformation Agrobacterium VirE2 protein binds to single-stranded DNA (Gietl et al, 1987;Christie et al, 1988;Citovsky et al, 1988Citovsky et al, , 1989Das, 1988) and protects transferred T-strands from nucleolytic degradation in the plant (Yusibov et al, 1994;Rossi et al, 1996). Because histone H2A-1 may also protect single-stranded T-DNA from degradation, we tested whether a HTA1 gene could compensate for loss of VirE2 during Agrobacterium-mediated plant transformation.…”

Section: Histone H2a-1 Does Not Increase Expression Of Previously Intmentioning

confidence: 99%

Overexpression of SeveralArabidopsisHistone Genes IncreasesAgrobacterium-Mediated Transformation and Transgene Expression in Plants

et al. 2009

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The Arabidopsis thaliana histone H2A-1 is important for Agrobacterium tumefaciens-mediated plant transformation. Mutation of HTA1, the gene encoding histone H2A-1, results in decreased T-DNA integration into the genome of Arabidopsis roots, whereas overexpression of HTA1 increases transformation frequency. To understand the mechanism by which HTA1 enhances transformation, we investigated the effects of overexpression of numerous Arabidopsis histones on transformation and transgene expression. Transgenic Arabidopsis containing cDNAs encoding histone H2A (HTA), histone H4 (HFO), and histone H3-11 (HTR11) displayed increased transformation susceptibility, whereas histone H2B (HTB) and most histone H3 (HTR) cDNAs did not increase transformation. A parallel increase in transient gene expression was observed when histone HTA, HFO, or HTR11 overexpression constructs were cotransfected with double-or single-stranded forms of a gusA gene into tobacco (Nicotiana tabacum) protoplasts. However, these cDNAs did not increase expression of a previously integrated transgene. We identified the N-terminal 39 amino acids of H2A-1 as sufficient to increase transient transgene expression in plants. After transfection, transgene DNA accumulates more rapidly in the presence of HTA1 than with a control construction. Our results suggest that certain histones enhance transgene expression, protect incoming transgene DNA during the initial stages of transformation, and subsequently increase the efficiency of Agrobacterium-mediated transformation. INTRODUCTIONAgrobacterium tumefaciens-mediated plant genetic transformation is an important experimental tool for investigation of various aspects of plant biology and for agricultural biotechnology. Development of this transformation technology represents the culmination of many decades of effort to improve tissue culture and plant genetic engineering techniques. Agrobacteriummediated transformation is based on a conjugative transfer-like process, which eventually takes the T-DNA to the host cell nucleus (Gelvin, 2000(Gelvin, , 2003a(Gelvin, , 2009Tzfira and Citovsky, 2003;Citovsky et al., 2007). After attachment of the bacterium to plant cells and induction of Agrobacterium virulence (vir) genes, a single-stranded DNA (the T-strand) is processed from the resident tumor-inducing or root-inducing plasmid and transported from the bacterium to the plant cell. In addition, a number of Virulence effector proteins, including VirD2 (attached to the T-strand), the single-strand DNA binding protein VirE2, plus VirE3, VirD5, and VirF are also transferred to the plant (Otten et al., 1984;Stahl et al., 1998;Vergunst et al., 2000Vergunst et al., , 2003Vergunst et al., , 2005Schrammeijer et al., 2003). These proteins are likely involved in protecting T-DNA from nuclease digestion, directing T-DNA to the nucleus, stripping proteins from the T-strand prior to integration, and integrating T-DNA into the plant genome. T-DNA integration occurs randomly into genomic DNA by the process of illegitimate recombination (...

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Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

Cited by 166 publications

References 36 publications

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

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

Recognition of the Agrobacterium tumefaciens VirE2 Translocation Signal by the VirB/D4 Transport System Does Not Require VirE1

Overexpression of SeveralArabidopsisHistone Genes IncreasesAgrobacterium-Mediated Transformation and Transgene Expression in Plants

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