Visualization of effector protein translocation from Agrobacterium tumefaciens into host cells

In our research protein translocation from Agrobacterium into yeast and plant cells is studied to obtain fundamental insights in the translocation process and in the fate of the translocated proteins in the host cells and the potential biotechnological applications of Agrobacterium mediated protein translocation were explored. In this thesis we studied the Agrobacterium virulence protein expression, translocation and localization via direct visualization and also potential biotechnological applications of protein translocation from Agrobacterium into the recipient cells. Plant science

Zinc Finger Artificial Transcription Factor-Mediated Chloroplast Genome Interrogation in Arabidopsis thaliana

The large majority of core photosynthesis proteins in plants are encoded by nuclear genes, but a small portion have been retained in the plastid genome. These plastid-encoded chloroplast proteins fulfill essential roles in the process of photochemistry. Here, we report the use of nuclear-encoded, chloroplast-targeted zinc finger artificial transcription factors (ZF-ATFs) with effector domains of prokaryotic origin to modulate the expression of chloroplast genes, and to enhance the photochemical activity and growth characteristics of Arabidopsis thaliana plants. This technique was named chloroplast genome interrogation. Using this novel approach, we obtained evidence that ZF-ATFs can indeed be translocated to chloroplasts of Arabidopsis plants, can modulate their growth and operating light use efficiency of PSII, and finally can induce statistically significant changes in the expression levels of several chloroplast genes. Our data suggest that the distortion of chloroplast gene expression might be a feasible approach to manipulate the efficiency of photosynthesis in plants

Increased Agrobacterium-mediated transformation of Saccharomyces cerevisiae after deletion of the yeast ADA2 gene

Agrobacterium tumefaciens is the causative agent of crown gall disease and is widely used as a vector to create transgenic plants. Under laboratory conditions, the yeast Saccharomyces cerevisiae and other yeasts and fungi can also be transformed, and Agrobacterium-mediated transformation (AMT) is now considered the method of choice for genetic transformation of many fungi. Unlike plants, in S. cerevisiae, T-DNA is integrated preferentially by homologous recombination and integration by non-homologous recombination is very inefficient. Here we report that upon deletion of ADA2, encoding a component of the ADA and SAGA transcriptional adaptor/histone acetyltransferase complexes, the efficiency of AMT significantly increased regardless of whether integration of T-DNA was mediated by homologous or non-homologous recombination. This correlates with an increase in double-strand DNA breaks, the putative entry sites for T-DNA, in the genome of the ada2Δ deletion mutant, as visualized by the number of Rad52-GFP foci. Our observations may be useful to enhance the transformation of species that are difficult to transform.Plant science

Zinc Finger Artificial Transcription Factor-Mediated Chloroplast Genome Interrogation In Arabidopsis thaliana

The large majority of core photosynthesis proteins in plants are encoded by nuclear genes, but a small portion have been retained in the plastid genome. These plastid-encoded chloroplast proteins fulfill essential roles in the process of photochemistry. Here, we report the use of nuclear-encoded, chloroplast-targeted zinc finger artificial transcription factors (ZF-ATFs) with effector domains of prokaryotic origin to modulate the expression of chloroplast genes, and to enhance the photochemical activity and growth characteristics of Arabidopsis thaliana plants. This technique was named chloroplast genome interrogation. Using this novel approach, we obtained evidence that ZF-ATFs can indeed be translocated to chloroplasts of Arabidopsis plants, can modulate their growth and operating light use efficiency of PSII, and finally can induce statistically significant changes in the expression levels of several chloroplast genes. Our data suggest that the distortion of chloroplast gene expression might be a feasible approach to manipulate the efficiency of photosynthesis in plants.Plant science

Visualization of effector protein translocation from Agrobacterium tumefaciens into host cells

In our research protein translocation from Agrobacterium into yeast and plant cells is studied to obtain fundamental insights in the translocation process and in the fate of the translocated proteins in the host cells and the potential biotechnological applications of Agrobacterium mediated protein translocation were explored. In this thesis we studied the Agrobacterium virulence protein expression, translocation and localization via direct visualization and also potential biotechnological applications of protein translocation from Agrobacterium into the recipient cells. </table