Research themes - Molecular and Developmental Genetics
- Research projects
B-NHEJ in plants: Identification of components involved in an alternative NHEJ pathway in plants
Qi Jia (CSC 2006-2010), Sylvia de Pater and Paul Hooykaas
In eukaryotic organisms DNA double strand breaks (DSB) are repaired via two mechanisms: homologous recombination (HR) and non-homologous end-joining (NHEJ). In animals and plants the latter one is the preferred pathway. Several components involved in the NHEJ pathway have been identified, among others Ku70/80, Mre11 and Ligase 4. In the past few years several reports showed that inactivation of these components in animal cells does not completely abolish NHEJ activity. Mutational analysis and pharmacological approaches showed that poly(ADP-ribose) polymerase-1 (Parp1) and the XRCC1/Ligase III complex are involved in a back-up NHEJ pathway (B-NHEJ) in mammalian cells.
In plants, orthologs of NHEJ components have been identified. Mutation of these components results in hypersensitivity to radiation and chemicals that induce DSBs. These components also have a function in T-DNA integration in Agrobacterium-mediated transformation. However, in NHEJ T-DNA insertion mutants, random T-DNA integration still occurs, although at a lower frequency. This means that an alternative pathway for the repair of DSBs, is responsible for the residual integration of foreign DNA. This alternative pathway is possibly related to the B-NHEJ pathway identified in mammalian cells. The objective of the project is to identify components and protein complexes of the alternative NHEJ pathway in Arabidopsis and to inactivate this DNA repair route.
Functional analysis of the hAT-like protein Daysleeper
Marijn Knip (ALW 2007-2011), Sylvia de Pater and Paul Hooykaas.
A large fraction (up to 90% in some plants) of eukaryotic genomes consists of transposable (mobile) elements. These elements have played a very important role in the evolution of genomes. Besides polyploidy and segmental duplication, transposon amplification resulted in increased DNA content. Another important function of mobile elements has been the creation of new genes with important cellular functions. These mobile elements may capture genomic fragments, as has been described for Mutator-like DNA elements (MULEs) resulting in so-called ‘Pack-MULEs’. Several reports show the recycling of coding material of the transposons themselves for genetic innovation. Two recent reports describe the presence of human chimeric proteins containing SET histone methyltransferase domains and Mariner transposase, which appear to be involved in important biological processes.
In Arabidopsis the hAT-like transposase DAYSLEEPER is essential for plant development and influences directly or indirectly the expression of many genes. Since DAYSLEEPER has DNA binding activity it may function as transcription factor or regulator of chromatin structure. Our objective is to determine the function of DAYSLEEPER in plant development. The expression pattern (developmentally and anatomically) and cellular localization will be determined, in vitro and in vivo DNA binding sites will be identified and interaction with other proteins will be analyzed.
Recombination an old and new tool for plant breeding
Sylvia de Pater (Recbreed, EU 2009-2013), Bert van der Zaal and Paul Hooykaas.
Historically, plant breeding has relied upon the selection of desirable variants resulting from sexual hybridization between varieties, and occasionally from rare spontaneous inter-species hybridizations. In modern, and possibly in future breeding, genetic variation from a broader source, namely, beyond the species gene pool, can be exploited, by homologous recombination (HR) or by transgenesis. All these breeding tools are still inefficient and must be improved.
The RECBREED project will provide plant breeders with new tools allowing better control over HR in both somatic and meiotic cells. The expected outcomes of the proposed research are efficient gene targeting technologies for precise engineering of plant genomes as well as the ability to control rates of meiotic recombination between homologous or homeologous chromosomes in classical breeding. Our group is involved in the work package, which aims at enhancing HR through targeted double strand break (DSB) induction. DSBs will be induced by zinc-finger nucleases that can be custom-designed for target sequences anywhere in the genome.
Novel zinc finger-based strategies for elucidating and controlling homologous recombination in plants
Bert van der Zaal and Paul Hooykaas
In the last few years, zinc finger (ZF) technology has gained enormous attention within the field of functional genomics. The technology is based upon the availability of well-characterized 30 amino acid ZF moieties, each with a cognate 3 base pair DNA binding site, combined with the possibility to generate multi-fingered (polydactyl) PZF domains by fusing individual ZF domains. Recently, we have been first in describing that libraries of different ZF-based artificial transcription factors (ZF-ATFs) can be used to discover novel mutant phenotypes in Arabidopsis. The technique, also called “genome interrogation”, can be regarded as a brute force approach to see whether or not a particular genome can be triggered to reveal a novel desirable trait, in our case a mutant with an extremely high frequency of homologous recombination (HR). In order to discover the genes that are truly responsible for the phenotype of interest, these genes will have to be identified among a much larger number of genes that are also differentially regulated by the causal ZF-ATF, but not related to the phenotype. In fact, this conundrum is associated with all genetic traits where a transcriptional regulator – either endogenous or artificial - acts as a master switch for a metabolical or a developmental pathway: non-specific and non-causal events blur any useful insight into the triggered mechanism of interest. By further exploiting the possibilities for modulating the DNA binding domain of ZF-ATFs, we want to demonstrate that such standstills can be broken, in this case by providing an unprecedented level of understanding of HR in plants.
Studies of the virulence protein VirD2 of Agrobacterium tumefaciens with the aim to develop novel methods for site-directed mutagenesis in plants.
Maartje van Kregten (LU 2006-2010), Bert van der Zaal and Paul Hooykaas
It was recently discovered in our lab that the VirD2 protein of Agrobacterium, which is involved in T-DNA processing and is also covalently attached to the 5’-end of single stranded T-DNA, can tolerate N- and C-terminal fusions with other protein domains. These unexpected findings will be further exploited to gain fundamental insights in VirD2 functions. Despite the crucial importance of VirD2 for processing and transfer of T-DNA, the precise role of the C-terminal domain for T-DNA integration and the possible importance of a DNA-ligase function of VirD2 during T-DNA integration is a matter of debate. The importance of the C-terminal domain of D2 for T-DNA transfer and integration will be investigated in combination with assessing the ability of a VirF C-terminal domain to complement any deleterious effects of VirD2 deletions. Hybrid molecules of D2 domains fused with the VirF transfer domain will delineate the minimally required VirD2 regions for T-DNA processing. As a consequence of these investigations, it will also be possible to explore the use of VirD2 hybrid proteins as novel tools for site-directed mutagenesis (SDM) in higher plants by equipping VirD2 with nuclease domains that might aid in insertion of T-DNA at a predetermined genomic site.
Unravelling the hidden secrets of the master of genetic engineering, the bacterium Agrobacterium tumefaciens
Martijn Rolloos (TOP-CW 2007-2011), Suzanne Wolterink (TOP-CW 2008-2011), Philippe Sakalis (TOP-CW 2008-2012), Bert van der Zaal, Paul van der Heusden and Paul Hooykaas.
The soil bacterium Agrobacterium tumefaciens is capable of transferring part of its tumor-inducing (Ti) plasmid, the T-DNA, to eukaryotic cells, where it stably integrates into the host genome. The Agrobacterium-mediated transformation (AMT) is so popular because of its exceptional efficiency and precision. Why this is the case and why Agrobacterium has this unique ability for interkingdom DNA transfer is only partially understood. An important distinguishing feature of the Agrobacterium system is that it delivers a single stranded (ss) DNA molecule with at the 5’end the pilot protein VirD2 into host cells through a type4 secretion system (TFSS). The nuclear localization sequence (NLS) in VirD2 guarantees a rapid translocation into the nucleus of the host cells. Besides the T-strand-VirD2 complex, the TFSS is used also to secrete separately a set of virulence proteins into the host cells. The latter are effector proteins that aid in the transformation process. For instance the VirE2 effector protein is an ssDNA binding protein which coats (and thus protects) the T strand on its way to the nucleus. Host factors also play an important role in AMT: some of these are instructed by the Agrobacterium effector proteins and play an important role in T-DNA integration in the host genome. A number of (conserved) host factors important for AMT have been identified by us and by others in the past. So far however a systematic survey of the host factors involved in AMT has not been done.
In this proposal we bring together three projects which are meant to obtain a more complete picture of this transformation process by using yeast functional genomics to study AMT. We shall use the collection of yeast deletion mutants to complete the survey for host proteins involved in AMT. Preliminary results from our lab suggest that chromatin-remodeling factors may play an important role in T-DNA integration besides the NHEJ-proteins identified before. In the first project we shall focus on the role of histone modification and chromatin remodeling in AMT. In the second project we shall
focus on the pilot protein VirD2, study its role in T-DNA integration and its possible interaction with chromatin factors. In the third project we shall try to follow effector protein delivery into host cells by real time confocal microscopy and study the function of VirD5, which was recently identified as a novel effector protein in our lab.
The role of protein ubiquitination in Agrobacterium-mediated transformation of Saccharomyces cerevisiae.
Xiaolei Niu (CSC 2007-2011), Paul van Heusden en Paul Hooykaas
During Agrobacterium-mediated transformation of eukaryotic cells a number of virulence proteins are transferred into the host cell. One of these proteins is VirF. VirF is an F-box protein which is thought to be involved in the targeted proteolysis of another virulence protein (VirE2) thus uncoating the T-DNA complex allowing efficient integration into the host genome. In this project we want to explore the function of VirF protein in Agrobacterium-mediated transformation in more detail using the yeast S. cerevisiae as model organism.
Modelling the role of 14-3-3 proteins in cation homeostasis.
Wouter Hendriksen (ALW/SYSMO 826.06.004 2007-2010) and Paul van Heusden
14-3-3 proteins form a family of highly conserved proteins present in all eukaryotic tissues investigated. These proteins can bind more than 300 different intracellular proteins. In this way, the 14-3-3 proteins regulate the activity of enzymes, regulate the subcellular localization of proteins and stimulate protein-protein interactions. These activities are important for many cellular processes like apoptosis, the cell cycle, stress response and signal transduction. 14-3-3 proteins are related to a number of human diseases like cancer and neurological diseases as Parkinson’s disease, the Miller-Dieker syndrome and Alzheimer’s disease and are used in a diagnostic test for BSE (mad cow disease). We use the yeast Saccharomyces cerevisiae as model organism to study fundamental aspects of 14-3-3 proteins. This organism has two genes encoding 14-3-3 proteins, BMH1 and BMH2. As in higher eukaryotes, the S. cerevisiae 14-3-3 proteins are involved in many cellular processes and many different binding partners have been identified.
We participate in the SYSMO (SYStems biology of MicroOrganisms) project Translucent (‘Gene interaction networks and models of cation homeostasis in Saccharomyces cerevisiae’; http://www.sysmo.net/index.php?index=61). 14-3-3 proteins may regulate cation homeostasis at different levels as they bind to relevant transcription factors, regulate protein levels and bind to proteins known to regulate ion transporters. We will investigate the role of 14-3-3 proteins in cation homeostasis.
The plant compass: dynamic AGC kinase signalling complexes that orient development by directing auxin transport.
Myckel Habets (CW-TOP 2010-2014), Yuanwei Fan (CSC 2009-2013), Fang Huang (CW-TOP 2009-2010), Remko Offringa
The plasticity of plant development allows plants to adapt to sudden changes in their environment. This plasticity is nicely illustrated by a plant’s growth toward the direction of light or gravity, and also by the unique ability of plant tissues or even single cells to regenerate into a new plant. A major determinant of plant development is the polar intercellular transport of the plant hormone auxin (indole-3-acetic acid), which regulates cell division and cell expansion by establishing dynamic auxin maxima and -gradients. Polar auxin transport is driven by the PIN transporter-like membrane proteins, whose asymmetric sub-cellular localization determine the direction of transport.
The group, with a team of international collaborators, has shown that the PINOID (PID) protein kinase determines the cellular polarity of the PIN auxin transporters. Further research has uncovered interacting/regulatory (KIR) proteins of PID and of the related PID-like kinases. Based on these findings we hypothesize that the combined action of PID, the PID-like kinases and the KIR proteins provides plants with a compass that integrates both external and internal signals to direct auxin transport, thereby determining plant growth orientation and architecture. The objective of this project is to test the plant compass hypothesis and, in a broader sense, to examine potential paradigms underlying the molecular mechanisms governing cell polarity in both plants and animals.
The role of plant AGC kinases in vascular development
Xiong Yang (KNAW 2009-2011 ), Remko Offringa
An important step in the evolution of higher land plants is the development of vascular tissues that allow efficient long-distance source to sink transport of nutrients and metabolites, and at the same time provide physical support for upward growth. Plant vascular development is of considerable economic importance, as it is a primary determinant of crop plant architecture and stature, and it is the source of wood formation in tree trunks. The plant hormone auxin is one of the primary signals in vascular development. Its polar transport generates auxin maxima that provide positional cues for many developmental processes, such as vascular patterning. The directional auxin flow depends on the activity of PIN auxin efflux carriers, whose polar subcellular localization is regulated in Arabidopsis by the PINOID (PID) protein kinase. PID is a membrane-associated AGC protein kinase that directs PIN polarity by determining the phosphorylation status of the PIN central hydrophilic loop. This project aims to investigate the role of PID and other AGC kinases, and their interacting proteins, in vascular patterning in Arabidopsis.
New tools and strategies for fruit breeding in vegetable crops
Myckel Habets(STW LPB6822, 2008-2010), Adam Vivian-Smith, Remko Offringa
Fruit set and -retention and parthenocarpy (seedlessness) are agriculturally important traits. The manipulation of fruit set and retention can improve crop yield, while introduction of the quality trait of parthenocarpy into fruit and vegetable crops enhances edibility through seed elimination. Previous research has shown that the plant hormone auxin is an important primary signal in these processes.
In this project we develop new breeding strategies for parthenocarpy in sweet pepper, the most important greenhouse vegetable crop in the Netherlands. In contrast to existing strategies – our new strategies are based on endogenous regulators of fruit set, thus allowing improved fruit set and/or parthenocarpy through a non-GMO approach. We employ genetic and genomics tools available in Arabidopsis to identify endogenous regulators for fruit set. Mutagenesis approaches will be developed for sweet pepper, enabling those trait loci that are involved in parthenocarpy in Arabidopsis (and other species) to be identified and captured for improvement of sweet pepper.
A polar auxin transport model as a novel breeding tool for shoot branching traits in crops
Kees Boot (TTI-GG 2CC-036RP 2009-2011), Kees Libbenga, Bert Peletier, Sander Hille, Bert van Duijn, Remko Offringa
The optimization of plant architecture is a common theme in breeding of crop and ornamental plants, and has resulted in the Green Revolution in the world’s food production between the 1940s and 1960s with branched cereal crops of reduced height as a prime result. Basipetal polar auxin transport through a plant’s stem is a central driver for branching. The scope of this project is to perform experiments designed to validate a new quantitative convection-diffusion model of polar auxin transport through Arabidopsis inflorescences, and to subsequently use this model as a tool to understand branching phenotypes in crop plants. The principal objectives of this project are:
1. To map the transporting tissues, and to obtain a better understanding of the basipetal auxin transport via the plant stem, through a reiterative process of experimentation and mathematical modeling.
2. To test whether this polar auxin transport-based model allows us to make predictions on shoot branching in the model plant Arabidopsis.
3. To identify sweet pepper lines from a mutant population based on their auxin transport profile and branching phenotypes.
4. To use the model and our knowledge of Arabidopsis to study branching in sweet pepper, using new and existing lines with different branching characteristics.