Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the... Show moreAgrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Pol theta)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Pol theta. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Pol theta in T-DNA integration. Pol theta deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants. Show less
The type-IV secretion system (T4SS) is a machinery able to transfer DNA and proteins between bacteria and in certain cases also to eukaryotic cells. This thesis compared the T4SSs present in... Show moreThe type-IV secretion system (T4SS) is a machinery able to transfer DNA and proteins between bacteria and in certain cases also to eukaryotic cells. This thesis compared the T4SSs present in Agrobacterium tumefaciens and the conjugative plasmid RP4. A. tumefaciens is able to transfer DNA into plant cells and transform them genetically. We discovered that a novel hairpin structure on the terminus of the transferred DNA allows for earlier expression of its genes in the recipient plant cell. Further, we could describe the translocation signal of the relaxase protein TraI of RP4 responsible for DNA processing and transfer via the T4SS. Additionally, it was shown that the T4SS of RP4 is able to translocate proteins from one cell to another without parallel DNA transfer. Furthermore, we were able to create a hybrid relaxase created consisting of parts from A. tumefaciens (relaxase part) and RP4 (translocation signal) that was shown to be translocated via the T4SS of RP4. Show less
The type-IV secretion system (T4SS) is a machinery able to transfer DNA and proteins between bacteria and in certain cases also to eukaryotic cells. This thesis compared the T4SSs present in... Show moreThe type-IV secretion system (T4SS) is a machinery able to transfer DNA and proteins between bacteria and in certain cases also to eukaryotic cells. This thesis compared the T4SSs present in Agrobacterium tumefaciens and the conjugative plasmid RP4. A. tumefaciens is able to transfer DNA into plant cells and transform them genetically. We discovered that a novel hairpin structure on the terminus of the transferred DNA allows for earlier expression of its genes in the recipient plant cell. Further, we could describe the translocation signal of the relaxase protein TraI of RP4 responsible for DNA processing and transfer via the T4SS. Additionally, it was shown that the T4SS of RP4 is able to translocate proteins from one cell to another without parallel DNA transfer. Furthermore, we were able to create a hybrid relaxase created consisting of parts from A. tumefaciens (relaxase part) and RP4 (translocation signal) that was shown to be translocated via the T4SS of RP4. Show less
The Gram-negative soil bacterium Agrobacterium tumefaciens is capable of infecting a large number of dicotyledonous plants, causing crown gall disease. During infection, a small DNA segment (T-DNA... Show moreThe Gram-negative soil bacterium Agrobacterium tumefaciens is capable of infecting a large number of dicotyledonous plants, causing crown gall disease. During infection, a small DNA segment (T-DNA) from the tumor-inducing (Ti) plasmid of the bacterium is delivered via a VirB/D4 Type Four Secretion System (TFSS) into plant cells where it is integrated into the genome. The expression of genes on the T-DNA in transformed plant cells leads to uncontrolled cell division, and ultimately triggers plant tumor formation. Agrobacterium also transfers several virulence (vir) proteins (VirD2, VirD3, VirD5, VirE2, VirE3 and VirF) encoded by genes present in the vir region of the Ti plasmid to host cells via the same TFSS apparatus independently of the T-DNA. In this thesis, I mainly focused on the biological functions of one of these translocated virulence proteins called VirD5. Show less
Agrobacterium tumefaciens is a phytopathogen which is known to be the causal agent of crown gall disease in dicotyledonous plants. Virulent Agrobacterium strains are always carrier of a tumor... Show moreAgrobacterium tumefaciens is a phytopathogen which is known to be the causal agent of crown gall disease in dicotyledonous plants. Virulent Agrobacterium strains are always carrier of a tumor-inducing (Ti) plasmid. As a result of the expression of Vir proteins, a section of this plasmid termed the T-DNA is transferred via a type four secretion system (T4SS) as a T-strand to the recipient cell where it can integrate into the genomic DNA of the recipient cell. In this thesis the prerequisites of T-DNA circle formation using yeast as a model were assayed. It was found that the homologous repair (HR) protein Rad52 is of importance to the formation of T-DNA circles. In addition to this, the effects of double strand break (DSB) induction on the efficiency of T-DNA integration via the HR pathway in yeast and the role the nucleosome occupancy of the target locus has on DSB induction were assayed. It was found that a decrease in the nucleosome occupancy of a target locus had no measurable impact on the efficiency of T-DNA integration at this locus. However, the combined translocation of nuclease proteins aimed at the target locus and T-strands during AMT greatly facilitated HR-mediated integration of T-DNA. Show less
Agrobacterium tumefaciens is a gram-negative soil bacterium that induces plant tumors by transferring a segment of DNA, called T-DNA, into plant cells. Under laboratory conditions, Agrobacterium... Show moreAgrobacterium tumefaciens is a gram-negative soil bacterium that induces plant tumors by transferring a segment of DNA, called T-DNA, into plant cells. Under laboratory conditions, Agrobacterium can also transform many different non-plant organisms such as the yeast Saccharomyces cerevisiae. During this process, a number of virulence proteins, including VirF and VirE3, are translocated into the host cell. VirF contains an F-box domain and, according to current theory, in plants and in yeast may induce degradation of the virulence protein VirE2 and the transcription factor VIP1, required for the integration of the T-DNA into host chromosomal DNA. VirE3 functions as a potential plant transcriptional activator. In our study, we expressed the Agrobacterium virulence proteins VirF and VirE3 in Saccharomyces cerevisiae and Arabidopsis thaliana and studied the effect of virF and virE3 expression on the genome-wide transcription in S. cerevisiae and A.thaliana using DNA microarrays and RNA-sequencing. Show less
Agrobacterium tumefaciens is a pathogenic bacterium, which can genetically transform plants and other organisms. It does so by translocating a part of its DNA, the T-strand, in complex with the... Show moreAgrobacterium tumefaciens is a pathogenic bacterium, which can genetically transform plants and other organisms. It does so by translocating a part of its DNA, the T-strand, in complex with the relaxase protein VirD2. We have shown that VirD2 is the determinant of translocation of the VirD2-T-strand complex, rather than the T-strand. Furthermore, we have elucidated the role of the DUF domain of VirD2. DUF is shown to be important for localization of VirD2 to the cell poles. Polar localization is essential for translocation of VirD2 to the recipient cell. Using VirD2 as a carrier, we have shown that several biotechnological important enzymes can be transferred to Arabidopsis thaliana. Of the relaxase-homing endonuclease fusion protein VirD2-I-SceI, we have shown that it can induce the formation of double-strand breaks in the Arabidopsis genome after translocation from Agrobacterium. Show less
