Sylvia de Pater
I am interested in plant DSB repair and Agrobacterium T-DNA integration with the ultimate aim to improve the rather low frequency of gene-targeting in plants.
My research interest is DSB repair and Agrobacterium T-DNA integration in plants, which knowledge will be ultimately applied to optimize site-specific mutagenesis and the rather low frequency of gene-targeting. Several research lines are being followed: 1) inactivation of random integration and 2) enhancing homologous recombination by introducing specific double strand breaks using custom-made endonucleases. To prevent unwanted DNA integrations, nucleases are also being introduced as proteins via the type four secretion system (T4SS) or as T-DNA in plant mutants that fail to integrate T-DNA.
I worked on several projects with the aim of improving the rather low frequency of gene-targeting in plants. We follow two approaches: inactivation of random integration and enhancing homologous recombination by introducing specific double strand breaks and increase the number of available repair emplates. For this latter approach we made use of zinc-finger nucleases (ZFN) and Tal Effector nucleases (TALEN). More recently, we focus on the use of CRISPR/Cas nucleases. In addition we try to further enhance gene-targeting by using cells that have a more active homologous recombination machinery.
Furthermore, methods are being explored for targeted mutagenesis (also called “genome editing”) of plant genes via a non-GMO approaches, using site-specific nuclease proteins expressed in Agrobacterium and transported via its T4SS to plant cells or through transformation of plant mutated in the Polθ gene that no longer integrate T-DNA. After transfer, the nucleases will induce double strand breaks (DSBs) at a specific location in the genome, which upon imperfect repair via non-homologous end joining (NHEJ) might result in mutations.
T-DNA integration and DNA Repair.
Closely related to the above subject I study the different DNA repair pathways in plants. In eukaryotic organisms DNA double strand breaks (DSB) are repaired via two mechanisms: homologous recombination (HR) and non-homologous end-joining (NHEJ). I want to identify the different components of these repair pathways in order to manipulate the balance between them. Furthermore repair pathways are being studied responsible for integration of Agrobacterium T-DNA in the host genome. A essential protein is Polymerase θ, which action together with other important factors, leads to the integration of the T-DNA in the plant genome.
- Hexi Shen (2017) Role of non-homologous end-joining in T-DNA integration in Arabidopsis thaliana.
- Marijn Knip (2012) DAYSLEEPER: from genomic parasite to indispensable gene.
- Qi Jia (2011) DNA repair and gene-targeting in plant end-joining mutants.
- Sandra Langeveld (2001) Role of reversibly glycosylated polypeptides in starch biosynthesis.
- Flip Hoedemaeker (1995) Structure and stability of legume lectin.
- Ron van Eijsden (1994) Mutational analysis of pea lectin.
I am manager of the greenpoint facilities at the ground floor and the tissue culture facilities at the fifth floor and I supervise the supporting staff involved (Ward de Winter and Jan Vink).
I am chair of the Education Committee Biology.
I am teacher at the second year course Molecular Biology and the Master course Advanced Molecular Genetics and Biotechnology.
BSc and MSc students are welcome to do an internship under my supervision on one of the subjects described under research. The subjects can be very divers with many different techniques; therefore, the program will be made just before the start of the internship. Possible subjects concern DNA repair pathways in plants and improvement of gene-targeting by using custom-made nucleases. Techniques may be used like cloning, plant transformation, analysis of transgenic plants and mutants on DNA, RNA and proteins level.