The RNA guided endonuclease based on the CRISPR/Cas system of Streptococcus pyogenus is a potent new tool for genome engineering in plants. Delivery of this system is usually done by... Show moreThe RNA guided endonuclease based on the CRISPR/Cas system of Streptococcus pyogenus is a potent new tool for genome engineering in plants. Delivery of this system is usually done by transforming plants (and other organisms) with Agrobacterium tumefaciens which transfers a T-DNA encoding the required genes through its Type IV Secretion System (T4SS). Instead, here we report the delivery of the Cas9 protein of the CRISPR/Cas system of Streptococcus pyogenus through the T4SS of Agrobacterium to Nicotiana benthamiana and Saccharomyces cerevisiae. Show less
The development of methods for the genetic modification of plants a few decades ago has provided a tremendous boost for molecular plant science. Crop plants have been generated that are... Show moreThe development of methods for the genetic modification of plants a few decades ago has provided a tremendous boost for molecular plant science. Crop plants have been generated that are resistant to insects or herbicides, or that produce useful sugars or healthy nutrients. Although the ban on growing GM crops in Europe has considerably limited the application of GM technologies, they have still contributed considerably to fundamental plant science. Especially by using the natural and very efficient mechanism of DNA transfer by the soil born bacterium Agrobacterium tumefaciens, many collections of mutant lines of model plant species such as Arabidopsis and rice have been generated, in which genes are disrupted or overexpressed by the insertion of an Agrobacterium transfer DNA (T-DNA) construct. These collections have been used in forward or reverse genetics studies to unravel the function of a gene or a family of genes in plant defense or development, and to identify the key regulators in these processes. The study described in this thesis focused on the use of one of these key regulators, the Arabidopsis AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 15/REJUVENATOR (AHL15/RJV), to alter developmental processes such as flowering, senescence and regeneration. Show less
Agrobacterium is the natures genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium... Show moreAgrobacterium is the natures genetic engineer that can transfer genes across the kingdom barriers to both prokaryotic and eukaryotic host cells. The host genes which are involved in Agrobacterium-mediated transformatiom (AMT) are not well known. Here, I studied in a systematic way to identify the whole eukaryotic host genes which are involved in this event. For this, I used a collection of around 4800 homozygous diploid deletion mutants and the cDNA libraries of yeast Saccharomyces cerevisiae as the eukaryotic host and two strains of Agrobacterium. First, a large-scale AMT protocol for the transformation of yeast mutants was developed. Then, the yeast mutant collection was screened for the mutants with an increased or a decreased AMT. 249 deletion mutant strains were found which affect AMT. Of great importance were host chromatin remodeling and histone modifying complexes. Besides, using GFP technique I showed the nuclear localization of Agrobacteriums VirD2 in yeast. Yeast two-hybrid screens also identified 12 yeast putative interaction partners of VirD2 protein. Show less
