Thesis opportunities of the Laboratory of Molecular Biology

Here you will find all the thesis projects from the Laboratory of Molecular Biology. Interested? Please contact us.

Plant architecture and development (Van Esse Group)

My group’s research focuses on understanding the genetic and molecular networks that are control different yield related traits such as developmental timing, seed and tiller number. We use natural and induced variation to study the genetic loci that control these key traits and state of the art ~omics tools to place these genes in precise molecular and cellular context. Our long term goal is to build a solid fundamental understanding of the genes and molecular networks that control plant architecture and development.

We make use of sophisticated 'omics'-technologies, including ChIPseq, RNAseq, yeast n-hybrid assays, and CRISPR mutagenesis. We welcome, on regular basis, BSc and MSc students that join our research team and perform their own project affiliated to ongoing research. Students are supervised by enthusiastic and highly motivated PhD students and researchers. Below some examples of the various opportunities for Bsc and Msc students.

Interested? Please contact:

Topics:

CRISPR-Cas mutagenesis and cereal development

CRISPR-Cas mutagenesis and cereal development

Targeted mutagenesis is a important part in our research to unravel the role of key regulatory genes in plant development. Within our laboratory, we use on routine basis CRISPR-Cas mutagenesis approaches in various plant species. Within this team, we mainly focus on CRISPR-Cas in barley.

Typical student projects involve construct design, using Golden Gate cloning, for targeted mutagenesis. Depending on the length of the thesis and project availability, you will also gain experience with plant transformation to perform genome editing and analyse the mutations/deletions created.

Interested gaining experience with CRISPR during your BSc or MSc thesis project? Please feel free to contact us to discuss the various opportunities.

Protein-Protein and Protein-DNA interaction studies

On regular basis, we perform protein-protein and protein-DNA interaction studies in our team using yeast-n-hybrid assays, but also other complementary techniques such as Co-immunoprecipitations. Typical questions raised are for example, what protein-protein interaction does a key regulator have? Or where does this regulator bind to the DNA?

Interested in protein-protein or protein-DNA interactions? Please feel free to contact us to discuss opportunities within the ongoing projects.

DNAprotein.jpg

Bioinformatics

Within our team, we have on regular basis bioinformatic data sets to be analysed. This can range from Transcriptional profiling (RNA-Seq) to natural variation studies, phylogenetics, variant-calling, and detailed protein-domain analysis.  

Interested in doing a bioinformatics BSc or MSc thesis project? Please contact us to discuss the various opportunities.

Understanding the role of gene duplications in cereals- CRISPR

FLOWERING LOCUS-T (FT) is a key flowering time activator that acts antagonistically to CENTRORADIALIS (CEN), which acts as a inhibitor of flowering. Both FT and CEN are crucial determinants of flowering time.

FT/CEN-like gene family members are characterized by extensive gene duplication events that occurred independently in nearly all flowering plants, which has led to diversification in the roles of these genes resulting in sub-and neofunctionalization: In potato FT-like genes are important regulators of tuberization; and in wheat TaFT2 influences spikelet development and floret fertility, while this gene only shows minor effects on flowering time.  The FT/CEN gene family exemplifies that based on sequence similarity alone, it is thus hard to understand the function of a gene crop species, especially when gene duplications have arisen.

Figure 1. The FT/CEN-like, regulatory FD TF and interacting TCP TF family members in barley and Arabidopsis. These gene families are highly diverse and have more members in barley when compared to the plant dicot model Arabidopsis. FT/CEN are members of the plant phosphatidylethanolamine binding protein (PEBP) family, which is divided into three main clades, FLOWERING LOCUS T (FT)-like, TERMINAL FLOWER1 (TFL1)-like, and MFT-like.
Figure 1. The FT/CEN-like, regulatory FD TF and interacting TCP TF family members in barley and Arabidopsis. These gene families are highly diverse and have more members in barley when compared to the plant dicot model Arabidopsis. FT/CEN are members of the plant phosphatidylethanolamine binding protein (PEBP) family, which is divided into three main clades, FLOWERING LOCUS T (FT)-like, TERMINAL FLOWER1 (TFL1)-like, and MFT-like.

Within this thesis project the student will focus on understanding the role of gene duplications and the impact on barley plant architecture and development.

Typical student gains experience with:

  • CRISPR in barley, including construct design
  • Tissue culture
  • Golden Gate cloning
  • PCR, genotyping
  • Project/time management, writing and presenting your thesis

Interested? Contact PhD student:

Unravelling genetic networks that control tiller and spike development in barley

Cereals such as barley and wheat are important crop species for food and feed supply. Cereal crop yield depends on traits such as the number of seeds per spike, the number side shoots (tillers) and the seed weight. These traits are often negatively correlated which makes improvement of yield difficult. Recent evidence indicates that the same genes affect both seed and tiller number. Optimizing yield by changing these traits is possible, but will require an in-depth and fundamental understanding of the molecular mechanisms that affect both inflorescence and shoot branching. The regulation of branching has been well studied in model systems like Arabidopsis and rice. In comparison, little is known about the regulation of branching in temperate cereals such as barley and its close relative wheat.

Within this project, we will study the molecular bases of tiller and/or inflorescence (spike) development in barley.

Figure 1. Barley development. During vegetative development the barley main shoot apex (MSA) is located at the basal internode of the plant called the crown. Developing leafs are surrounding the MSA and additional side shoots called tillers may be formed in the leaf axils. After transition from the vegetative to the reproductive development the MSA moves upwards and differentiates into the inflorescence. The seeds on the barley plant are located at the inflorescence (spike). The spike architecture can be arranged as two-rowed or six-rowed. In the two-rowed cultivars only the central spikelet is fertile while in the six-rowed cultivars all three spikelets produce seeds.
Figure 1. Barley development. During vegetative development the barley main shoot apex (MSA) is located at the basal internode of the plant called the crown. Developing leafs are surrounding the MSA and additional side shoots called tillers may be formed in the leaf axils. After transition from the vegetative to the reproductive development the MSA moves upwards and differentiates into the inflorescence. The seeds on the barley plant are located at the inflorescence (spike). The spike architecture can be arranged as two-rowed or six-rowed. In the two-rowed cultivars only the central spikelet is fertile while in the six-rowed cultivars all three spikelets produce seeds.

Typical student gains experience with:

  • Y2H screen Protein-protein interactions
  • Phylogenetic analysis
  • Conserved domain analysis using natural allelic variation
  • Transfer knowledge obtained in barley to other crops
  • Selection CRISPR-CAS9 mutagenized plants in barley
  • Project management, writing and presenting your thesis

Cereal root development: What happens below ground?

Within this project, which is a collaboration between the van Esse and Willemsen group we are unravelling the impact of PLETHORA gene family members on cereal root development. For this, we use a scale of different techniques, but also CRISPR mutagenesis and generation of marker lines for detailed phenotyping.

Typical student gains experience with one or more of the following topics:

Interested? Contact:

Stem cell specification and regeneration (Heidstra Group)

A central question in biology is what determines the fate of a cell, tissue or even organ. Fate decisions taking place during embryogenesis are reiterated during the life of the plant to generate the adult architecture. Starting point in our studies on fate specification is the model plant Arabidopsis thaliana and its anatomically simple root is.  

Within my team there is are often possibilities for BSc and MSc thesis projects. Below a some information on the diverse opportunities. Interested? Please don’t hesitate to contact us! 

Topics:

CRISPR-Cas in plant development

Recent advances with the RNA-mediated CRISPR-Cas systems have dramatically transformed our ability to specifically modify intact genomes of diverse cells and organisms. The CRISPR–Cas system has proven to be an efficient, simple, and robust gene-targeting technology with enormous potential for applications across basic science, agricultural and biotechnology. 

We have standardized the CRISPR-Cas technology for efficient use in Arabidopsis with the purpose to create in vivo specific mutations and deletions in the genome for genes involved in plant development. We target genes involved in stem cell biology, lateral root formation and cell division/differentiation. Particularly useful is the ability to perform multiplex CRISPR-Cas to either generate multiple mutations or to delete genes from the genome. 

You generate the different modules that are used to generate CRISPR-Cas construct. For this purpose you will get familiar with the modular Golden Gate cloning technology which presents a directional method to construct vectors carrying multiple inserts. Finally, you will perform genome editing and analyse the mutations/deletions created, both molecularly and phenotypically. 

Used skills

  • Standard recombinant DNA technology such as DNA isolation, PCR and (Golden Gate) cloning.
  • Handling and transformation of model organisms; E.coli, agrobacterium, arabidopsis.
  • Gene expression analysis by way of quantitative RT-PCR.
  • Analysis of gene knock-outs by way of PCR-genotyping and microscopy-phenotyping.

Requirements

  • For BSc thesis: MOB20306
  • For MSc thesis: MOB20306 and MOB30306 or MOB31303 or MOB30806 or PHP30806 or equivalent

SCHIZORIZA: tissues with multiple personalities

Asymmetric cell division is a fundamental and universal mechanism for generating diversity and pattern in multicellular organisms. SCHIZORIZA (SCZ), a transcriptional co-factor controls the separation of cell fate in a set of stem cells generating different Arabidopsis root tissues: root cap, epidermis, cortex, and endodermis. Loss-of-function, expression, and reconstitution experiments indicate that SCZ acts mainly from within its cortical expression domain in the stem cell niche, exerting both autonomous and non-autonomous effects to specify cortex identity and control the separation of cell fates in surrounding layers. 

The SCZ protein contains a DNA binding site but lack an transcriptional activation sequence whereas it does contain putative transcriptional repressor domains. Thus we want to determine to which proteins the SCZ can bind to form a transcriptional activator or repressive complex and what role these proteins play in plant development in general. In addition, we perform transcriptomics and DNA binding experiments to determine downstream target genes and determine their role in tissue specification. 

Used Skills

  • Standard recombinant DNA technology such as DNA isolation, cloning and PCR.
  • Handling of various model organisms like E.coli, Agrobacterium, yeast, Arabidopsis.
  • Yeast-2-Hybrid screening for SCZ interactors and confirm interactions in yeast.
  • BiFC (split-YFP) to test complex formation in planta using confocal laser microscopy.
  • Transient and stable plant transformation to investigated identified genes by way of promoter-reporter assays in planta. Gene knock-out analysis, genotyping and phenotyping using microscopy and gene expression analysis.

Requirements

  • For BSc thesis: MOB20306
  • For MSc thesis: MOB20306 and and MOB30306 or MOB31303 or MOB30806 or PHP30806 or equivalent

Arabidopsis root patterning

PhD project of Renan Pardal.

Arabidopsis thaliana roots have a very characteristic pattern, with well defined and morphologically distinguishable tissue layers. SCZ (SCHIZORIZA) gene codes a HSF (Heat-Shock Transcription Factor) that was shown to be involved in the patterning of Arabidopsis thaliana roots.

Single point mutations in SCZ leads to drastic changes in the root morphology: the QC (quiescent centre) that is very clear in WT (wild type) roots is no longer visible, there is an extra endodermal layer (Figure 1A) and the cortical layer starts to develop root hairs, which in WT roots only appears on epidermal cells (Figure 1B). Additionally, analysis of cell identity markers shows that the identity of several tissues were mixed in mutant roots (Figure 1A), and more drastically, there is no longer a tissue layer expressing cortical markers. Altogether these results show that SCZ is necessary for the cortex specification and for the separation of other tissues identities. Through the use of different molecular techniques this project aims to uncover how SCZ is exerting its function on root patterning. Additionally, since SCZ codes a heat-shock transcription factor, another goal of this project is to investigate the role of SCZ in stress response. 

Some techniques employed in this project: Golden Gate and Gateway cloning, CRISPR-Cas9 genome editing, yeast-two-hybrid interaction assay, RNA-sequencing, in-situ hybridization and confocal microscopy. 

Arabidopsis root patterning

Stem cell maintenance in the Arabidopsis root

All tissues are ultimately generates by the activity of stem cells. In the root of the model plant Arabidopsis a group of stem cells surround a mitotically inactive organizer, named Quiescent Center (QC). Both the activities of the parallel acting transcription factors PLETHORA (PLT) and SCARECROW (SCR) are required for the specification and function of the QC, and thereby the maintenance of the surrounding stem cell. Consequently, the respective mutants display short roots with loss of stem cells and consumed meristem. We have isolated the target genes of PLT2 and SCR specifically from the QC. Now we want to investigate the function of these genes regulated by PLT2 and SCR in the stem cell niche. 

You will confirm the regulation of target genes, generate and analyze promoter-reporter fusions, and investigate the corresponding knockouts for phenotype. Together, these results should identify additional genes in the network required for root stem cell niche function.

Used skills

  • Standard recombinant DNA technology such as DNA isolation, PCR and (Golden Gate) cloning.
  • Handling and transformation of model organisms; E.coli, agrobacterium, arabidopsis.
  • Gene expression analysis by way of quantitative RT-PCR.
  • Design and construct promoter-reporter(GFP) fusions.
  • Analysis of promoter activity in planta by confocal laser microscopy.
  • Analysis of gene knock-outs by way of PCR-genotyping and microscopy-phenotyping.
  • Construction of double mutants by crossing and analysis of these by the above methods.

Requirements

  • For BSc thesis: MOB20306
  • For MSc thesis: MOB20306 and and MOB30306 or MOB31303 or MOB30806 or PHP30806 or equivalent

How many embryos fit in one seed?

Embryos in Arabidopsis are characterized by an embryo proper, which generates most of the mature embryo, that is connected to the maternal tissues by a suspensor. During normal embryo development it is believed that the embryo proper signals to the suspensor thereby suppressing the embryonic program in these cells. 

The meerling (mrl) mutant is polyembryonic, meaning that multiple embryos are formed in the seed. These embryos all generate normal looking healthy seedlings upon germination that grow out to become wild type looking plants. Unlike the few mutants known that can give rise to two (twin) embryos, the mrl mutant seeds can contain up to five embryos that originate from the sustained embryonic suspensor. 

We have identified the genetic defect in the mrl mutant and now we want to confirm the causal relationship between mutation and phenotype. You will further characterize mutant and the gene responsible. In addition, you will detail the phenotypic defects that originate during polyembryony by analysing marker gene expression to find out where and how it all goes wrong. Furthermore, the MRL gene is potentially regulated by a microRNA which you will further investigate. Finally, you will search for interactors of the MRL protein and test their putative involvement in polyembryony.

Used Skills

  • Standard recombinant DNA technology such as RNA/DNA isolation, PCR and (Golden Gate) cloning.
  • Handling and transformation of model organisms; E.coli, agrobacterium, yeast, arabidopsis.
  • Gene expression analysis by way of quantitative RT-PCR.
  • Design and construct promoter-reporter(GFP) fusions.Analysis of promoter activity in planta by confocal laser microscopy.
  • Yeast-2-hybrid (Y2H) screening and analysis of interactors.
  • Analysis of gene knock-outs by way of PCR-genotyping and microscopy-phenotyping.
  • Construction of double mutants by crossing and analysis of these by the above methods.

Requirements

  • For BSc thesis: MOB20306
  • For MSc thesis: MOB20306 and and MOB30306 or MOB31303 or MOB30806 or PHP30806 or equivalent

Regeneration genes

Introduction of transgenes in plants has been a force in molecular biology and biotechnology for decades. Agrobacterium tumefaciens is generally used as a vehicle to introduce genetic material in the plant cell. Unfortunately, not all plants (particularly agronomically important crops) have the ability to regenerate a complete plant from a single (transgenic) cell. 

Therefore, identification and knowledge on the molecular factors involved in the regeneration process is required [1]. To gain more information on the regeneration process, an RNA-sequencing experiment was conducted using Arabidopsis thaliana regenerating tissue at multiple time-points up to the fully regenerated shoot. 

In this project the goal is to uncover candidates whose function is currently not associated with the regeneration process, as well as characterize the differential expression of genes suspected to be involved in regeneration through time. This requires the analysis of the RNAseq dataset individually, comparing expression data from different time-points, and to existing datasets. Genes/transcripts can be clustered based on co-expression [2,3] measures to find genes that show expression patterns similar to regeneration related genes. Clustered genes can then be analysed for enriched Gene Ontology annotations or common transcriptional regulators [4] to learn more about the underlying regulatory mechanisms. 

References

1. Radhakrishnan D, Kareem A, Durgaprasad K, Sreeraj E, Sugimoto K, Prasad K: Shoot regeneration: a journey from acquisition of competence to completion. Current Opinion in Plant Biology 2018, 41:23-31. 

2. Langfelder P, Horvath S: WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 2008, 9:559. 

3. Serin EAR, Nijveen H, Hilhorst HWM, Ligterink W: Learning from Co-expression Networks: Possibilities and Challenges. Frontiers in Plant Science 2016, 7:444. 

4. Kulkarni SR, Vaneechoutte D, Van de Velde J, Vandepoele K: TF2Network: predicting transcription factor regulators and gene regulatory networks in Arabidopsis using publicly available binding site information. Nucleic Acids Research 2018, 46:e31-e31. 

Used Skills 

Genomics, programming, statistics, machine learning 

Requirements

BIF30806, MAT22306 or ABG30806 

Regeneration recalcitrance in plants 

PhD project of Jana Wittmer.

Regeneration recalcitrance is a problematic feature of several crop species, which prevents efficient scientific research in these crops. In this project you will attempt to obtain information on regeneration recalcitrance by using transformation and a unique regeneration method on several different crops. In addition, you will perform tissue culture experiments in Arabidopsis. Together, these data should yield greater insight in the mechanism of recalcitrance.

Used Skills

  • Standard recombinant DNA technology such as DNA isolation, PCR and (Golden Gate) cloning.
  • Handling and transformation of model organisms; E.coli, agrobacterium, Arabidopsis.
  • Gene expression analysis by way of quantitative RT-PCR.
  • Analysis of promoter activity in planta by confocal laser microscopy.
  • Tissue culture.
  • Handling of, and transformation in multiple crop species.Crossing and analysis of these by the above methods.

Requirements

  • For BSc thesis: MOB20306
  • For MSc thesis: MOB20306 and and MOB30306 or MOB31303 or MOB30806 or PHP30806 or equivalent

De novo meristem formation (Kohlen Group)

My team is interested in unravelling the underlying molecular-physiological mechanisms that control a plants ability to initiate new axes of growth. We aim to integrate transcriptional and metabolomic data to generate a transcriptional metabolomic network. Not only can such a network be observed over time, we can also use it to identify genes to target for manipulation. For this, we employ a wide range of different molecular, physiological and bioinformatical tools. These tools range from; CRISPR-CAS9 to mass spectrometry and from single cell sequencing to protoplasting and cell sorting (FACS). We are regularly looking for new students, both on the level of BSc. and MSc. In my view, a  thesis or internship can best be discussed and tailored to the specific needs of both the student and the research group. Do you find this research interesting? Would you like to contribute to our work and learn some state-of-the-art new techniques? If yes, feel free to drop me a line!

Molecular control of flowering and reproduction of plants (Immink Group)

Flowering induction marks the start of the reproduction cycle and the final reproductive success of plants strongly depends on the timing of this developmental phase transition. Plants integrate various environmental cues, such as daylength, light quality and temperature with endogenous signals to time flowering, seed set and seed germination. But how do different plant species, such as Arabidopsis, tulip, tomato and sugar beet adapt their reproduction cycle to environmental conditions and optimize their reproductive success? To study this, we make use of sophisticated 'omics'-technologies, including ChIPseq, RNAseq, yeast n-hybrid assays, and CRISPR mutagenesis. We aim at deciphering the molecular mode-of-action of key regulatory proteins, with special emphasis on transcription factors and chromatin modifying proteins. We have continuously BSc and MSc thesis projects available, associated to the above research questions and supervised by enthusiastic and highly motivated PhD students, post-docs or researchers.  

Interested? Please contact:

RNA Biology of Plant Embryos (Nodine Group)

MicroRNAs (miRNAs) are short non-coding RNAs that mediate target transcript repression in plants and animals. Although miRNAs are required throughout development, relatively little is known regarding their embryonic functions. To systematically characterize embryonic miRNAs in plants, we recently developed high-throughput sequencing based methods to profile hundreds of miRNAs and associated targets throughout Arabidopsis thaliana (Arabidopsis) embryogenesis (Schon et al., 2018, Genome Research; Plotnikova et al., 2019, Plant Cell). We discovered dozens of miRNAs that dynamically cleave and repress target transcripts including 30 that encode transcription factors.

These miRNA:target interactions have a profound impact on embryonic gene expression programs, and we demonstrated that the miRNA-mediated repression of several transcription factors were individually required for proper division patterns of various embryonic cell lineages (Fig. 1). These data indicate that the miRNA-directed repression of multiple transcription factors is critically important for the establishment of the Arabidopsis body plan.

Figure 1 | miRNA-Mediated Repression of Transcription Factors is Required for Morphogenesis   Example of how a miRNA localized to outer cells (left) restricts its target transcript factor to inner cell types (center) to enable proper embryo morphogenesis and developmental timing (right). (For more details, see Plotnikova et al. 2019, Plant Cell)
Figure 1 | miRNA-Mediated Repression of Transcription Factors is Required for Morphogenesis Example of how a miRNA localized to outer cells (left) restricts its target transcript factor to inner cell types (center) to enable proper embryo morphogenesis and developmental timing (right). (For more details, see Plotnikova et al. 2019, Plant Cell)

Excitingly, we have also discovered a dozen miRNA:target interactions that are highly enriched in embryos and appear to be involved in various processes including hormone signaling, organelle biology, pathogen defense and DNA methylation. For example, we have found that one miRNA dynamically cleaves and represses a transcript encoding a DNA methyltransferase during embryogenesis, and this is required to prevent the ectopic methylation of thousands of genes. By studying the dynamic activities of this single miRNA during embryogenesis, we have uncovered how it helps balance the high methyltransferase activity during early embryogenesis that is required to silence mutagenic transposable elements while preventing off-targeting (i.e. epigenetic collateral damage) of protein-coding genes.

Figure 2 | Small RNA-Mediated Establishment of Nascent Epigenome   Diagram illustrating how small RNAs dynamically help establish genome-wide DNA methylation patterns during embryogenesis. CHROMOMETHYLASE 3 (CMT3) levels are high during early embryogenesis and establish DNA methylation on transposons. During mid-embryogenesis, miR823 mediates the repression of CMT3 to prevent it from ectopically methylating protein-coding genes (Papareddy et al. 2021, bioRxiv). As CMT3 levels decrease, 24 nt small interfering RNAs (siRNAs) increase and guide de novo methyltransferases (e.g. DRM2) to transposons and establish methylation and subsequent silencing (based on Papareddy et al. 2020, Genome Biology). mCHH, CHH methylation (H ≠ G) guided by siRNAs/DRM2 and CMT2; mCHG, CHG methylation activities of CMT3.
Figure 2 | Small RNA-Mediated Establishment of Nascent Epigenome Diagram illustrating how small RNAs dynamically help establish genome-wide DNA methylation patterns during embryogenesis. CHROMOMETHYLASE 3 (CMT3) levels are high during early embryogenesis and establish DNA methylation on transposons. During mid-embryogenesis, miR823 mediates the repression of CMT3 to prevent it from ectopically methylating protein-coding genes (Papareddy et al. 2021, bioRxiv). As CMT3 levels decrease, 24 nt small interfering RNAs (siRNAs) increase and guide de novo methyltransferases (e.g. DRM2) to transposons and establish methylation and subsequent silencing (based on Papareddy et al. 2020, Genome Biology). mCHH, CHH methylation (H ≠ G) guided by siRNAs/DRM2 and CMT2; mCHG, CHG methylation activities of CMT3.

We have internship and thesis projects aimed at characterizing the functions of these embryo-enriched miRNA:target interactions further. We will use a combination of microscopy, genetics, genomics and additional methods to study embryo-enriched miRNA:target interactions further.

Molecular development of Arbuscular Mycorrhizal symbiosis (Limpens Group)

To live in environments where nutrients are limited, plants engage in an endosymbiosis with arbuscular mycorrhizal (AM) fungi. These fungi colonize plant roots and are hosted inside root cortex cells, where highly branched hyphal structures called arbuscules are formed (Figure 1). There, the fungi deliver scarce minerals, especially phosphate and nitrogen sources, that they take up from the soil to the plant for which they get sugars and lipids in return. The AM symbiosis is very ancient and occurs in the vast majority of all land plants, which makes it one of the agriculturally and ecologically most important endosymbioses in plants. 

Figure 1. Highly branching AM hyphae (stained green) forming an arbuscule inside a root cortex cell.
Figure 1. Highly branching AM hyphae (stained green) forming an arbuscule inside a root cortex cell.

Understanding how AM fungi are accommodated inside plant roots and how nutrient exchange is controlled is of major importance as it determines the symbiotic efficiency of the interaction. Therefore, we use molecular, genetic- and cell-biological approaches to study the molecular mechanisms by which AM fungi optimize symbiotic nutrient transfer; both from the plant and the fungal side.For more information of specific topics please contact me:

Thesis subjects can be done in the following projects:

How is arbuscule development regulated?

The ability to host AM fungi inside cells requires a reprogramming of root cortex cells, which is controlled by a signalling cascade initiated between fungus and plant. Several key factors controlling arbuscule formation, branching and maintenance (lifetime) have been identified. To unravel the process of arbuscule development we use cell-type specific (transcriptome) analyses to identify novel key regulators and their interactors. A major challenge will be to unravel how these factors work together to control arbuscule development. Currently, we are focussing among others on the role of communication between endodermis and cortex to control arbuscule development and the role of phosphate-sensing transcriptional regulators in the regulation of arbuscule lifetime.

In this research reverse genetic analyses (CRISPR/RNAi/transposon-tagged lines), confocal fluorescence microscopy, protein-protein interactions (co-IP, mass-spec, Y2H) and cell-specific transcriptome analyses (in situ, reporter constructs, qPCR and RNAseq) are used to get insight into the molecular processes that control arbuscule development.

arbuscule_cortexcell.JPG

How does genetic variation within fungal isolates contribute to symbiotic efficiency?

One of the key questions in AM biology is to understand at a molecular level what determines how much growth benefit the plant has from interacting with AM fungi. It has been shown that some fungi improve plant growth much more than others. Such variation is not only observed between AMF species or isolates of the same species but even within fungal individuals. However, the molecular basis for such functional diversity is still largely unknown. We try to study how genetic variation between nuclei that populate the common hyphal network (Figure 3) is distributed, their dynamics and ultimately how it affects plant performance. A major challenge for the future will be to find ways to genetically manipulate the fungus. 

In this project we combine next-generation sequencing with single cell, spore and single nucleus analyses. Nanotechnology will be pioneered (together with Viola Willemsen) to manipulate the fungus. In addition to wet lab experiments, the project also offers a possibility to focus more on bioinformatics analyses of next-gen sequencing data.

How does the hyphosphere microbiome influence AM symbiosis?

In this novel project, a with collaboration the Chinese Agricultural University (prof. Feng Gu and dr. Lin Zhang), we aim to study how AM fungi work together with bacteria in the soil to facilitate their interaction with the plant; ie. a tripartite interaction between plant-fungus-bacteria. It has become clear that AM fungi can recruit specific bacteria to their extraradical hyphae, which can facilitate the uptake of organic nutrients. We aim to study how bacteria are recruited, the influence of fungal or plant genotypes on which bacteria are recruited and what their role is in the efficiency of the AM interaction with the plant. 

In this project, we will use high throughput comparative DNA (amplicon) sequencing for identification of bacteria in combination with meta-transcriptome sequencing. Isolated bacteria will be characterized and their influence on fungus and plant will be studied depending on predictions from bioassays and transcriptome data. Additionally, the role of fungal effector proteins on hyphosphere composition will be studied.


Nodulation Engineering (Geurts Group)

The Nitrogen Fix – Few projects in plant biotechnology are harder, or promise a greater payoff than enabling crops to make their own nitrogen fertilizer.

The above title is a citation of a Science paper in 2016 (Stockstad, 2016, Science 353:12225-7) that reflected the massive expectations researchers have about engineering nitrogen-fixing root nodules on crops. Such root nodules are well known from legumes and are the subject of study for over a hundred years. However, not only legumes possess the trait. Outside the legume family, root nodules occur in 9 taxonomic lineages. Among these is Parasponia, a small genus in the Cannabis family, representing five tropical tree species.

Parasponia trees can form nitrogen-fixing root nodules with the same rhizobium bacteria that also can nodulate legume plants. Genomics and molecular genetic studies demonstrated that the nitrogen-fixing nodulation trait in legumes and Parasponia have a shared evolutionary origin. We use Parasponia as a comparative system to legumes to identify the critical genetic adaptations allowing plants to form nitrogen-fixing root nodules. Subsequently, this information is used to engineer crop plants, of which cassava is our main target.

Interested? Contact:

Topics:

Comparative bioinformatics to identify genes that correlate with nodulation trait 

By using a phylogenomic approach we discovered three critical genes that associate with nodulation. However, these genes may reflect only a tip of the iceberg. Ongoing genome sequencing initiatives allow high resolution comparisons of genomes and expression data of related nodulating and non-nodulating plant species. Besides gene discovery associated with symbiosis, we also aim to identify cis-regulatory elements regulating symbiotic genes that differ between nodulating and non-nodulating plants. In this way adaptations in nodulation genes and promotor regions can be discovered. 

In a research thesis you will come familiar with bioinformatic tools and methods such as comparative genomics, working on the Linux command line, and programming in R or Python. 

Image_Geurts_group_phyton.jpeg

Design-Built-Test of engineering constructs

To successfully engineer a complex genetic trait in crops, it is essential to have a fast and efficient cycle of designing engineering constructs, built these constructs, and test them in vivo. The design of novel gene constructs is guided by bioinformatic analysis of nodulating and non-nodulating plant species. An important tool in our engineering strategy also involves the design of efficient marker gene constructs for discrete steps in rhizobium-induced nodule formation. These include markers specific for rhizobium-induced signalling, developmental markers to discriminate between nodule and lateral root organogenesis, and markers for bacterial infection events and nitrogen fixation.

Newly designed constructs are built using synthetic biology and golden gate cloning. Subsequently, the functionality of the constructs is tested in legume models (e.g. Medicago truncatula and Lotus japonicus), the nodulating non-legume Parasponia, and non-nodulating sister species that have lost the nodulation in course of evolution. To do so, fast and efficient transformation protocols have been established, allowing a ‘design-built-test’ cycle within ~3 months.

In a research thesis you will come familiar with this ‘design-built-test’ cycle and all techniques which are associated with it.

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Engineering cassava

Cassava as an important carbon-rich staple food for millions of people in West-Africa and other regions in the world. We selected cassava as an engineering target crop because the species is phylogenetically closely related to the so-called nitrogen-fixation clade. The nitrogen-fixation clade represents all plant species that can form root nodules in symbiosis either with rhizobium bacteria or Frankia bacteria. Current hypothesis on root nodulation in the nitrogen-fixing clade suggests a single evolutionary origin. The cassava ancestor just missed this opportunity!

We established a fast Agrobecterium rhizogenes-mediated root transformation protocol for cassava. This protocol allows bypassing the tedious Agrobacterium tumefaciens transformation procedure that is generally used for cassava. By using the A. rhizogenes transformation protocol, cassava plants carrying transgenic roots can be obtained in 4 weeks, allowing fast testing of engineering constructs.

In a research thesis you will come familiar with cassava biology, root development, transformation and engineering strategy.

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Plant architecture in relation to the environment (Willemsen Group)

Within my team we study key cellular and developmental mechanisms in plants. Our group investigates pattern formation, cell polarity, cell cycle, growth and regeneration, also in response to mutualistic microbes. We use model, non-model and crop plants. We combine molecular genetic, genomic and biochemical approaches with bioinformatics and computational modelling of biological processes to answer relevant research questions in plant cell and developmental biology. 

Below, some details on ongoing projects:

How succession shapes adaptation of natural populations of Arabidopsis thaliana 

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In spite of recent advances in molecular biology and ecology, we still do not understand mechanistically how plant life history strategies are shaped by environmental factors. We want to figure out how plant-soil interactions influence adaptation of these strategies. Our aim is to unravel how soil development during secondary succession influences the selection of plant life history strategies.

We are studying natural populations of Arabidopsis thaliana and how they are influenced by successional changes in biotic-abiotic soil conditions in a well-described time series of abandoned ex-arable fields. During secondary succession, the plant community shifts from pioneer to later succession species under influence of changes in both the abiotic and biotic composition of the soil. We have the hypothesis that belowground changes contribute to adaptation of plant roots to their specific soils, as roots are the interface of the plant and the soil. In this research, we are integrating soil ecology and molecular plant biology studying genomes, expression patterns, plant-soil feedback effects and root architecture. These findings will provide fundamental knowledge on root traits of crops adapted to sustainable soil management in agriculture.

  1. Studying the effect of soil succession on natural Arabidopsis populations
  2. Coupling genetic variation to soil succession to identify micro-evolution
  3. Investigating the role of root architecture in soil succession

Main techniques:

  • Standard molecular biological tools such as DNA isolation, PCR and sequencing
  • CRISPR-Cas9 mutagenesis and Golden Gate modular cloning
  • Arabidopsis transformation, genotyping and phenotyping
  • Confocal Laser Scanning Microscopy (CLSM)
  • Analysing root systems using the rhizotron system
  • Field work collecting natural Arabidopsis populations and succession soil
  • Large-scale plant-soil feedback experiments
  • Bioinformatics concerning Arabidopsis WGS and RNAseq as well as microbiome sequencing

Interested? Contact:

Unravelling the role of PLT transcription factors during early embryogenesis

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Embryogenesis is a crucial developmental process in flowering plants. The initial asymmetric division of the plant zygote produces different apical and basal daughter cells and has been linked with differential auxin distribution but the underlying mechanism for this generation of cell fate diversity remains unknown. Expression of the PLETHORA (PLT) genes, encoding AP2 domain containing stem cell transcription factors, is detectable from the earliest stages of embryogenesis onward. Two members of these plant specific transcription factor family are crucial at the first stages of embryogenesis. The function of PLT genes in early embryogenesis will be investigated. And we will try to isolate and analyse factors which are activating these important transcription factors.

Techniques your are going to use in this project are:

  • Standard molecular biological tools: (gate way)cloning, PCR, DNA isolation, sequencing, gel electrophorese, protein expression in bacteria, protein isolation, PAGE
  • Yeast-one-hybrid
  • Yeast-two-hybrid
  • EMSA
  • Confocal scanning microscopy
  • In Situ Hybridisation
  • Mutant analysis
  • Bioinformatics

Interested? Contact:

Linking molecular biology and ecology: Investigating the role of root architecture in soil succession

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In this project, we want to study the importance of root architecture for successful soil colonization thereby connecting molecular and plant developmental biology to terrestrial ecology. I am studying natural population of Arabidopsis thaliana derived from secondary succession fields. We want to test whether a specific root system architecture is optimal for growth in soils of certain succession stages. To do so, we are able to simplify and elaborate root architecture of Arabidopsis through well-established genetic and molecular knowledge. This can done by mutating lateral root, adventitious root and root cap developmental processes. 

In the project, you would be involved in generating the genetic constructs needed to change the root architecture, characterization of these root architecture mutants and to actually test these plants on different succession stage soils. Additionally, you would be screening root systems of natural Arabidopsis populations coming from these succession soils and check for phenotypic differences. 

Techniques you are going to use in this project are:

  • Standard molecular biological tools such as DNA isolation, PCR and sequencing
  • CRISPR/Cas9 mutagenesis and Golden Gate modular cloning
  • Arabidopsis transformation, genotyping and phenotyping
  • Confocal Laser Scanning Microscopy (CLSM)
  • Analysing root systems using the rhizotron system
  • Depending on season; field work collecting natural Arabidopsis populations and succession soil

Interested? Contact:

Plant Developmental Systems (PDS, Angenent Group)

Within the PDS group we are interested in how developmental processes are controlled by transcription factors and chromatin modifications. We aim to unravel transcriptional networks underlying various processes such as flowering time regulation, floral organ development, fruit formation and embryogenesis.  

For general questions about our research contact:

Transcription factors in Flower Development

Transcription factor complexes controlling flower development and their target genes

Flower development is one of the best understood and economically most important developmental processes in plants. It serves as a model system to understand organ specification and cellular differentiation, starting from pools of undifferentiated ‘stem cells’ residing in meristems. Flower development is controlled by transcription factors of the MADS-box family. These proteins act as major developmental switches to specify the identity of floral meristems and floral organs. They supposedly act in larger protein complexes that bind to the promoters of target genes and regulate them in an organ-specific fashion.

We want to understand how and why MADS protein complexes control the expression of their target genes in different floral organs and at different stages during flower development. The goal of this project is to identify MADS-box target genes that are dynamically regulated during development based on the results of genome-wide DNA-binding and expression studies. The expression dynamics of these target genes will be studied using fluorescent reporter gene studies (e.g. GFP). Binding of MADS-domain proteins and protein complexes will be studied by chromatin immunoprecipitation (ChIP) in planta and/or by Electrophoretic Mobility Shift Assays (EMSAs) in vitro.

Methods 

Gateway cloning, plant transformation, Confocal microscopy (CLSM), chromatin immunoprecipitation, Realtime quantitative PCR, Electrophoretic Mobility Shift Assays (EMSAs).

Interested? Contact:

Spatiotemporal expression dynamics of MADS-box transcription factors in flower development

Flower development in Arabidopsis is controlled by transcription factors of the MADS-box family. These proteins act as major developmental switches to specify the identity of floral meristems and floral organs. According to the current model of flower development, MADS-box transcription factors interact in a combinatorial fashion to specify the different types of floral organs, and to control organ differentiation and growth. MADS proteins are expressed in a highly cell-type specific and temporally dynamic fashion in developing flowers.

The goal of this project is to characterize and quantify MADS protein expression levels during flower development at the cellular level using fluorescent reporter gene analysis. These data will be used for modelling approaches to understand regulatory interactions within the MADS-box transcription factor network that are essential for flower formation.

Methods

Handling Arabidopsis - genotyping, making crosses, phenotypic analysis. Confocal microscopy (CLSM), CLSM data analysis. 

Interested? Contact:


Other topics:

Embryogenesis

In vitro embryogenesis

Plants are developmentally-plastic organisms. Not only do they continually differentiate new organs from the stem cell niche throughout their lifespan, but they also regenerate organs and even embryos during in vitro culture. We study different in vitro embryogenesis systems to gain a deeper understanding of how differentiated cells switch developmental pathways to form embryos in vitro. These include somatic embryogenesis, induced from vegetative plant cells by the synthetic auxin 2,4-D or by the BABY BOOM (BBM) and LEAFY COTYLEDON1 (LEC1) transcription factors, and embryogenesis induced from immature pollen grains or egg cells by stress, by inhibition of chromatin regulatory proteins or by BBM/LEC1. Our model systems include arabidopsis, Brassica napus and tomato. Identification of cis-regulatory elements We aim to identify cis-regulatory elements that control BBM and LEC1 expression in planta. We identify highly conserved promoter regions by phylogenetic footprinting and then mutate them using CRISPR-Cas9 mutagenesis. Reporter analysis, yeast one-hybrid analysis, mutant analysis and transient expression techniques are then used to correlate conserved promoter motifs with regulatory functions and specific DNA binding proteins.  

Molecular control of haploid embryogenesis 

Haploid cells like pollen grains can be converted into embryos in vitro. We aim to understand this process by identifying the changes that take place at the chromatin and gene expression levels as cells are induced toward embryogenesis. We study how tissue culture-induced changes in chromatin architecture and histone modifications activate or repress specific developmental pathways at the gene expression level. Much of this work is done on cell-type specific populations collected by flow sorting.  

Techniques

ChIP-seq, mRNA-seq, ATAC-seq, FACS, qRT-PCR, yeast n-hybrid screens, mutant analysis, CRISPR mutagenesis, Gateway and Golden Gate cloning, reporter analysis, confocal/DIC microscopy, chemical genomics, tissue culture, transformation. 

Interested? Contact:

Auxin and haploid embryo induction 

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Plants are very special because they can continually differentiate new organs from the stem cell niche throughout their lifespan, but they are also capable of regenerating new cell types and organs after wounding or from explants in tissue culture. 

Microspore embryogenesis is a unique process that beautifully illustrates this developmental plasticity of plant cells. In this process, the immature male gametophyte (microspore) is induced by a simple and short heat stress treatment to form haploid embryos in culture. The haploid embryos that arise can be germinated and converted to homozygous doubled haploids (DHs). Microspore embryogenesis is therefore widely used as a means to generate homozygous plants in a single generation, allowing plant breeders to accelerate their breeding programs. However, despite the practical advances of this process, the (epi)genetic and molecular mechanisms underlying this process remain poorly understood. 

The phytohormone auxin, which is essential for correct tissue patterning and organ formation, seems to play an early and important role in microspore embryogenesis: we observed the presence of an auxin response shortly after the heat stress treatment and this auxin response was specifically present in those cells that will eventually form haploid embryos in culture. Based on our current knowledge, we hypothesize that microspore embryogenesis is a two-step process comprising i) a stress‐related event needed for the switch from pollen development to embryo development, followed by ii) an endogenous auxin‐related event required for cell proliferation. 

This project aims to examine the role of stress and early auxin signaling during microspore embryogenesis. To this end, we will use different fluorescent auxin reporters in combination with transcriptome analysis to identify the stress- and auxin-related events during microspore embryogenesis. Additionally, we use novel and existing auxin compounds to identify the specific role of this hormone in microspore embryo induction. The overall focus is to improve our understanding of the molecular basis of microspore embryogenesis.

Used skills

Requirements

Good theoretical and practical basis in (plant) molecular biology.

Interested? Contact:

Regulation of tomato fruit growth and ripening

Phenotype of T0 mutants generated by using CRISPR/CAS9. rin single mutant (left), nor and rin double mutant (middle) and wild type Moneyberg (right)
Phenotype of T0 mutants generated by using CRISPR/CAS9. rin single mutant (left), nor and rin double mutant (middle) and wild type Moneyberg (right)

Fruits provide humans with essential nutrients for health benefits, such as vitamins, dietary fiber and minerals. Tomato is the model species for studying fleshy fruit development and ripening. 

We study transcription factors RIPENING INHIBITOR (RIN), NON- RIPENING (NOR) and COLORLESS NON-RIPENING (CNR), which are the main factors controlling ripening, together with gaseous hormone ethylene in tomato fruits. Functions of RIN, NOR and CNR have been known already through the study of naturally occurring mutants, but how they work together in a network to control colour, flavour and texture during fruit ripening by regulating many common downstream effector genes, is poorly understood. We aim to unravel the mechanisms of combinational action of tomato transcription factors on the expression of relevant ripening-related genes through: 

  • Studying the effector genes of combining different gene dosages of the major regulators on fruit ripening using heterozygous progeny of different crossed wild type and mutant parents (using natural mutations as well as our own generated CRISPR/CAS9-mutants)
  • Studying downstream gene promoter architecture using targeted deletion with CRISPR/CAS9

We are experienced in editing genome by using CRISPR/CAS9 and are continuing to develop new strategies in mutagenesis. Null mutants in genes encoding major transcription factors have been obtained and promoter mutagenesis experiments by multiple guide-RNAs are ongoing.

Used skills

  • Bioinformatics tools
  • Molecular cloning and sequencing
  • Protein (-DNA) interaction studies with yeast 1, 2- or 3- hybrid assays
  • Gene expression analysis with qRT-PCR
  • CRISPR/CAS9 mutagenesis and mutant characterization and phenotyping
  • Plant transformation and tissue culture

Requirements

Good theoretical and practical basis in (plant) molecular biology.

Interested? Contact:

Evolution of transcription factor function

Transcription factors (TFs) act together in complexes to regulate the expression of downstream target genes. Developmental processes such as flowering and fruit development are regulated by networks of transcription factor complexes: Gene Regulatory Networks (GRN). MADS-domain TFs play a major role in the GRNs that regulate flowering and fruit development. Within the flowering plants, a huge variety of inflorescences, flowers and fruits has evolved, induced by changes in the underlying GRNs. We are investigating the role that MADS-domain TFs play in the evolution of these traits and also use this for crop research. Currently, we focus on different aspects of flowering, comparing the functions of the MADS-box genes in Arabidopsis and tomato. This involves both in vitro and in planta techniques like yeast two-hybrid, yeast one-hybrid, EMSA, cloning and construct generation, CRISPR/CAS and plant transformation. 

Interested? Contact:

Evolution of FRUITFULL 

FRUITFULL (FUL) is a MADS-domain transcription factor that plays a major role in the development of the Arabidopsis fruit, the silique. In ful mutants, the silique remains very small and does not open to disperse the seeds. 

The evolution of plant species largely depends on the evolution of transcription factors. In this project, we are investigating the evolution of the transcription factor FRUITFULL, which is present as a single copy in the model species Arabidopsis, and regulates many important developmental processes such as leaf development, plant architecture, flowering time and fruit development. In tomato, there are four FRUITFULL genes that can each regulate a subset of these functions. 

To understand the evolution of the four tomato FRUITFULL proteins, we are investigating their properties and try to find out to which DNA sequences they can bind, which protein-protein interactions they form, what their functions are in tomato, and to what extent they can rescue the Arabidopsis fruitfull mutant. This information will also help us to dissect the functions of FRUITFULL in Arabidopsis, and to predict what functions FRUITFULL genes from other species will have. 

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Practical work 

The following experiments are performed to elucidate the evolution of the tomato FRUITFULL genes:

  • Yeast two-hybrid experiments to investigate the protein-protein interactions. Try to identify the amino acids that are responsible for changes in these interactions and perform the experiments with mutated proteins. (Cloning of constructs, PCR, yeast transformation, yeast two-hybrid).
  • DIP-Seq experiments to investigate to which DNA-sequence motifs the different FRUITFULL homologs can bind. (Recombinant protein expression, DIP-seq, high-througput sequencing, bioinformatics)
  • CRISPR mutagenesis to knock-out the genes in tomato and study the function of the different homologs. (Cloning constructs, tomato transformation, tissue culture, DNA-extraction, PCR, phenotyping)
  • Transformation of Arabidopsis fruitfull mutants with the tomato genes to determine to what extent they can rescue the phenotype. (Cloning constructs, Floral dip transformation, DNA-extraction, PCR, phenotyping).
  • Expression analysis of the tomato genes. (qPCR, cloning of reporter constructs).

Interested? Contact:

Physiology of seed quality

Seeds are the basis of most crop production systems. Poor seed quality results in poor crop establishment, waste of inputs, lower yields and farmers income. Less well known is that seed vigour also influences the sensitivity to pathogens. We study how we can improve seedling tolerance to pathogens by improving seed vigour, as part of the solutions in reducing or even omitting chemical crop protection. Carrot seeds and Alternaria pathogens are our present model system. 

Interested? Contact: