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VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an omega mutation. In addition, a mutant VirD2 protein lacking the omega domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the omega and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the omega domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

Somatic embryos of Arabidopsis thaliana can be produced from explants of developing zygotic embryos. Cultivation of explants on maturation medium results in development of three main classes of regenerative structures: adventitious shoots, fused shoots, and complete somatic embryos. The ontogeny and anatomy of these structures was examined using serial plastic sections. Furthermore, two molecular markers were assayed to monitor transcriptional auxin responses and formation of a root meristem in this process: the LENNY allele of PIN4, a transposon insertion creating a fusion to the reporter gene GUS; and DR5::GUS, a synthetic reporter of auxin-induced transcription. In zygotic embryogenesis, PIN4 expression is confined to the center of the root meristem and begins to be detectable by the globular stage of embryogenesis, while DR5::GUS expression marks an "auxin perception maximum" in the more distal regions of the root. Adventitious and fused shoots develop no anatomically recognizable root meristem and do not express either of the two markers at their basal pole. Instead, the vasculature of their axis is directly connected to the vasculature of the explant. By contrast, complete somatic embryos were only loosely attached to the explant, had an anatomically defined root meristem and showed expression of both markers at their root pole. Our results suggest that the establishment of a root meristem in somatic embryos required appropriate auxin levels during the course of their development.

We developed novel plasmids and T-DNA binary vectors that incorporate a modified and more useful form of the superpromoter. The superpromoter consists of a trimer of the octopine synthase transcriptional activating element affixed to the mannopine synthase2# (mas2#) transcriptional activating element plus minimal promoter. We tested a superpromoter-bglucuronidaseA fusion gene in stably transformed tobacco (Nicotiana tabacum) and maize (Zea mays) plants and in transiently transformed maize Black Mexican Sweet protoplasts. In both tobacco and maize, superpromoter activity was much greater in roots than in leaves. In tobacco, superpromoter activity was greater in mature leaves than in young leaves, whereas in maize activity differed little among the tested aerial portions of the plant. When compared with other commonly used promoters (cauliflower mosaic virus 35S, mas2#, and maize ubiquitin), superpromoter activity was approximately equivalent to those of the other promoters in both maize Black Mexican Sweet suspension cells and in stably transformed maize plants. The addition of a maize ubiquitin intron downstream of the superpromoter did not enhance activity in stably transformed maize.The availability of convenient vectors harboring a strong promoter that is active in all or most cells of different plant species would be useful for a variety of applications in plant molecular biology. We previously described a novel synthetic promoter consisting of a trimer of the octopine synthase (ocs) transcriptional activating element (ocs activator) linked to the mannopine synthase2# (mas2#) activator-promoter region (Ni et al., 1995). Initial studies in tobacco (Nicotiana tabacum) indicated that this promoter, called the superpromoter, could direct expression of GUS activity to a level 2-to 20-fold higher than the commonly used enhanced double cauliflower mosaic virus (CaMV) 35S promoter (Ni et al., 1995). The activity of the superpromoter was highest in roots, but also was high in leaves and stems.The superpromoter was originally created by ligating three ocs activator fragments (positions 2333 to 2116 relative to the transcription start site [Leisner and Gelvin, 1988]) from the ocs gene to the mas2# activator-promoter region (2318 to 165 relative to the transcription start site [Ellis et al., 1984]), all from the Agrobacterium tumefaciens Ti-plasmid pTiA6. The construction of the original superpromoter resulted in the repeated presence within the promoter of several commonly used restriction endonuclease sites (BamHI, EcoRI, HindIII), as well as the presence of the restriction endonuclease sites PstI and XhoI. This feature precluded easily linking genes to the promoter. In addition, it complicated further analysis of T-DNA insertions in the plant genome. We therefore modified the superpromoter, eliminating most of these internal restriction endonuclease sites. This modified superpromoter (MSP) formed the basis for the construction of several novel plant expression and T-DNA binary vectors. Here, we describe these vectors...

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