No abstract

The VirD2 protein of Agrobacterium tumefaciens was shown to pilot T‐DNA during its transfer to the plant cell nucleus. We analyze here its participation in the integration of T‐DNA by using a virD2 mutant. This mutation reduces the efficiency of T‐DNA transfer, but the efficiency of integration of T‐DNA per se is unaffected. Southern and sequence analyses of integration events obtained with the mutated VirD2 protein revealed an aberrant pattern of integration. These results indicate that the wild‐type VirD2 protein participates in ligation of the 5′‐end of the T‐strand to plant DNA and that this ligation step is not rate limiting for T‐DNA integration.

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumorinducing (Ti) plasmid, to the plant cell nucleus. The Tiplasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plaht cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein ofA. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.

Agrobacterium tumefaciens is able to transfer a piece of DNA, the T-DNA, to the nucleus of the plant cell. The VirD2 protein is required for the production of the T-DNA, it is tightly linked to the T-DNA and it is thought to direct it to the plant genome. Two nuclear localization signals (NLS), one in the N-terminal part and one in the C-terminal part of the VirD2 protein, have been shown to be able to target marker proteins to the plant nucleus. Here we analyze nuclear entry of the T-DNA complex using a new and very sensitive assay for T-DNA transfer. We show that optimal T-DNA transfer requires the VirD2 NLS located in the C-terminal part of the protein, whereas mutations in the N-terminal NLS coding sequence seem to have no effect on T-DNA transfer.

A General Surveillance plan to monitor for unanticipated adverse effects of genetically modified organisms (GMOs) on human health and the environment is required as part of the EU legislation for imported and cultivated GMOs.Imported GM products: Operators involved in the import, handling and processing of grain commodities entering the EU have monitoring procedures in place to survey products and are organised in associations across the EU Member States. They can therefore be considered as the best placed to monitor for potential unanticipated adverse effects linked to imports of GM products into the EU. Recognising this, the Plant Biotechnology Industry has established a collaboration to cover General Surveillance of grain commodity imports with the relevant associations, which is coordinated by EuropaBio, the European Association for Bioindustries.Cultivated GM plants: Monitoring for unanticipated adverse effects should take place in agronomic zones representative of commercial GM crop cultivation. Farmers are therefore considered to be the most valuable sources of information when it comes to General Surveillance of GM crop cultivation, due to their extensive experience with and direct involvement in cultivation. Acknowledging this, the Plant Biotechnology Industry has developed a harmonised general surveillance approach based on farmer questionnaires.

Applications for placing on the market of genetically modified organisms (GMOs) for import, food, feed and processing under Directive 2001/18/EC and Regulation (EC) No 1829/ 2003, have to include a monitoring plan conforming with Annex VII to Directive 2001/18/EC. One aspect of this monitoring plan is the need for general surveillance to identify the occurrence of adverse effects of the viable GMO or its use on human and animal health or the environment which were not anticipated in the environmental risk assessment (e.r.a.). Since international grain commodity trade consists of commingled products and the Plant Biotechnology Industry is not directly involved in commodity trade, authorisation holders under Directive 2001/18/EC and Regulation (EC) No 1829/2003 have been working together within the European Association of Bioindustries (EuropaBio) and with European trade associations representing relevant commodity trade operators to develop a harmonised general surveillance methodology for import and processing of viable GMOs. A harmonised industry general surveillance system was agreed upon by the Plant Biotechnology Industry members and the European trade associations and has been operational for several years. This harmonised industry general surveillance system has now been described in a harmonised industry monitoring plan. In line with Decision 2002/811/EC establishing guidance notes supplementing Annex VII to Directive 2001/18/EC and the Guidance Document of the Scientific Panel on Genetically Modified Organisms for the risk assessment of genetically modified plants and derived food and feed, this monitoring plan contains a detailed description of the agreed monitoring methodology together with other specifics, such as the baseline and controls for general surveillance, the time period over which general surveillance will be carried out, the use of existing networks and how the results of monitoring will be reported and reviewed.

Transferred DNA (T-DNA) is transferred as a single-stranded derivative from Agrobacteium to the plant cell nucleus. This conclusion is drawn from experiments expoiting the different properties of single-and double-stranded DNA to perform extrachromosomal homologous recombination in plant cells. After transfer from Agrobacterium to plant cells, T-DNA molecules recombined much more efficently if the homologous sequences were ofopposite polarity than ifthey were of the same polarity. This observation reflects the properties of single-stranded DNA; single-stranded DNA molecules of opposite polarity can anneal directly, whereas inglestranded DNA molecules of the same polarity first have to become double stranded to anneal. Judging from the relative amounts of single-to double-stranded T-DNA derivatives undergoing recombination, we infer that the T-DNA derivatives enter the plant nucleus in their single-stranded form. This results in tight attachment of VirD2 to a specific nucleotide on the 5' end of the lower strand of T-DNA. This leads to the production of three types ofT-DNA derivatives: circular double-stranded DNA (9-12), linear double-stranded DNA (13-15), and single-stranded DNA corresponding to the bottom strand of the T-DNA (16,17). While the circular form is produced in Agrobacterium at low quantities upon stimulation by plant inducers and is considered to be a by-product of the T-DNA complex formation (12, 18), linear single-and double-stranded T-DNA derivatives are produced at much higher levels and at similar rates (refs. 14, 15, and 19; for review, see ref. 6). Both linear single-and double-stranded T-DNA derivatives have been found tightly attached to VirD2 protein (14,15,(20)(21)(22). As the VirD2 protein was shown to pilot the T-DNA from the bacteria to the plant cell nucleus (23-27) and as it was suggested that VirD2 is also involved in integration (refs. ' (36, 37) and ofintermolecular homologous recombination ofT-DNAs containing sequence overlaps of the same polarity (38) and the finding of two T-DNAs integrated in a tail-to-tail configuration (39) point to the presence of free double-stranded T-DNA derivatives inside the plant cell nucleus.We designed experiments that could detect the transient (46). The essential difference between the two types of recombining partners, therefore, is that DNA molecules of opposite polarity shorten the recombination pathway by direct self-annealing, whereas those of the same polarity first must become double-stranded.' The rationale for the work described in this report was to exploit known properties of extrachromosomal recombination to construct T-DNA molecules whose recombination Abbreviations: T-DNA, transferred DNA; GUS, ,B-glucuronidase. 8000The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Agrobacterium tumefaciens causes neoplastic growth in plants by transferring a piece ofDNA, called T-DNA, into the nucleus of the plant cell. The virulence protein VirD2 ofA. tumefaciens is tightly linked to the T-DNA and is thought to direct it to the plant genome. Here we show that the VirD2 protein contains two nuclear localization signals that are functional both in yeast and in plant cells. One signal is located in the N-terminal part of the protein and resembles a singlecluster-type nuclear localization signal. The second signal is near the C terminus and is a bipartite-type nuclear localization signal. The involvement of these sequences in the entry of the T-DNA into the nucleus is discussed.Agrobacterium tumefaciens, the causative agent of crown gall disease, transfers into plant cells genes coding for enzymes involved in the synthesis of plant growth factors. These genes are carried by the T-DNA, a well-defined region of a large plasmid called Ti (tumor-inducing). The T-DNA is delimited at both extremities by two almost perfect 25-basepair repeats, or border sequences (for reviews see refs.