VirB/D4-dependent protein translocation from Agrobacterium into plant cells.

The Agrobacterium VirB/D4 transport system mediates the transfer of a nucleoprotein T complex into plant cells, leading to crown gall disease. In addition, several Virulence proteins must somehow be transported to fulfill a function in planta. Here, we used fusions between Cre recombinase and VirE2 or VirF to directly demonstrate protein translocation into plant cells. Transport of the proteins was monitored by a Cre-mediated in planta recombination event resulting in a selectable phenotype and depended on the VirB/D4 transport system but did not require transferred DNA.

Cre/lox-mediated recombination in Arabidopsis: evidence for transmission of a translocation and a deletion event.

Cre recombinase was used to mediate recombination between a chromosomally introduced loxP sequence in Arabidopsis thaliana (35S-lox-cre) and transferred DNA (T-DNA) originating from Agrobacterium tumefaciens (plox-npt), carrying a single loxP sequence. Constructs were designed for specific Cre-mediated recombination between the two lox sites, resulting in restoration of neomycin phosphotransferase (nptII) expression at the target locus. Kanamycin resistant (Km(r)) recombinants were obtained with an efficiency of about 1% compared with random integration. Molecular analyses confirmed that these were indeed due to recombination between the lox sites of the target and introduced T-DNA. However, polymerase chain reaction analysis revealed that these reflected site-specific integration events only in a minority (4%). The other events were classified as translocations/inversions (71%) or deletions (25%), and were probably caused by site-specific recombination between a randomly integrated T-DNA and the original target locus. We studied some of these events in detail, including a Cre-mediated balanced translocation event, which was characterized by a combination of molecular, genetic and cytogenetic experiments (fluorescence in situ hybridization to spread pollen mother cells at meiotic prophase I). Our data clearly demonstrate that Agrobacterium-mediated transfer of a targeting T-DNA with a single lox site allows the isolation of multiple chromosomal rearrangements, including translocation and deletion events. Given that the complete sequence of the Arabidopsis genome will have been determined shortly this method has significant potential for applications in functional genomics.

Cre/lox-mediated site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana by transient expression of cre.

The Cre/lox system was used to obtain targeted integration of an Agrobacterium T-DNA at a lox site in the genome of Arabidopsis thaliana. Site-specific recombinants, and not random events, were preferentially selected by activation of a silent lox-neomycin phosphotransferase (nptII) target gene. To analyse the effectiveness of Agrobacterium-mediated transfer we used T-DNA vectors harbouring a single lox sequence (this vector had to circularize at the T-DNA left- and right-border sequences prior to site-specific integration) or two lox sequences (this vector allowed circularization at the lox sequences within the T-DNA either prior to or after random integration, followed by targeting of the circularized vector), respectively. Furthermore, to control the reversibility of the integration reaction, Cre recombinase was provided transiently by using a cotransformation approach. One precise stable integrant was found amongst the recombinant calli obtained after transformation with a double-lox T-DNA vector. The results indicate that Agrobacterium-mediated transformation can be used as a tool to obtain site-specific integration.

Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase.

In this study Agrobacterium tumefaciens transferred DNA (T-DNA) was targeted to a chromosomally introduced lox site in Arabidopsis thaliana by employing the Cre recombinase system. To this end, Arabidopsis target lines were constructed which harboured an active chimeric promoter-lox-cre gene stably integrated in the plant genome. A T-DNA vector with a promoterless lox -neomycin phosphotransferase (nptII) fusion was targeted to this genomic lox site with an efficiency of 1.2-2.3% of the number of random events. Cre-catalyzed site-specific recombination resulted in restoration of nptII expression by translational fusion of the lox-nptII sequence in the integration vector with the transcription and translation initiation sequences present at the target site, allowing selective enrichment on medium containing kanamycin. Simultaneously, the coding sequence of the Cre recombinase was disconnected from these same transcription and translation initiation signals by displacement, aimed at preventing the efficient reversible excision reaction. Of the site-specific recombinants, 89% were the result of precise integration. Furthermore, approximately 50% of these integrants were single copy transformants, based on PCR analysis. Agrobacterium T-DNA, which is transferred to plant cells as a single-stranded linear DNA structure, is in principle incompatible with Cre-mediated integration. Nevertheless, the results presented here clearly demonstrate the feasibility of the Agrobacterium -mediated transformation system, which is generally used for transformation of plants, to obtain site-specific integration.

Analysis of nuclear and organellar DNA of somatic hybrid calli and plants between Lycopersicon spp. and Nicotiana spp.

Protoplast fusion experiments between Lycopersicon esculentum or L. peruvianum and Nicotiana tabacum or N. plumbaginifolia were performed to investigate the possibility of producing symmetric and asymmetric somatic hybrids between these genera. These fusions, which involved 1.7 x 10(8) protoplasts, yielded 35 viable hybrid calli. Plant regeneration was successful with two calli. One of these regenerants flowered, but developed no fruits. Analysis of the nuclear DNA by means of dot blot hybridization with species-specific repetitive DNA probes combined with flow cytometry, revealed that the nuclei of most hybrid calli contained the same absolute amount of Nicotiana DNA as the Nicotiana parent or (much) less, whereas the amount of Lycopersicon DNA per nucleus was 2-5 times that of the parental genotype. Eighteen of the 34 hybrids analyzed possessed Lycopersicon chloroplast DNA (cpDNA), whereas the other 16 had DNA from Nicotiana chloroplasts. The cpDNA type was correlated with the nuclear DNA composition; hybrids with more than 2C Nicotiana nuclear DNA possessed Nicotiana chloroplasts, whereas hybrids with 2C or less Nicotiana nuclear DNA contained Lycopersicon chloroplasts. Mitochondrial DNA (mtDNA) composition was correlated with both nuclear DNA constitution and chloroplast type. Hybrids possessed only or mainly species-specific mtDNA fragments from the parent predominating in the nucleus and often providing the chloroplasts. The data are discussed in relation to somatic incompatibility which could explain the low frequency at which hybrids between Lycopersicon and Nicotiana species are obtained and the limited morphogenetic potential of such hybrids.