Stem cell specification and regeneration (Heidstra Group)

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

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

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. 

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

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

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 

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