Student projects

Regulation of immunity signaling by cell surface receptors at the transcriptional level

Project description: Plants recognize pathogens via cell surface and intracellular immune receptors. In both cases, extensive transcriptional reprogramming occurs at early and late stages after the pathogen detection. However, the mechanisms of these processes are poorly understood. We have found that activation of the plasma membrane-bound receptor-like protein 23 (RLP23) by a pathogen-associated molecular pattern (PAMP) peptide nlp24 leads to massive gene expression reprogramming in Arabidopsis, which is dependent on the TOPLESS (TPL) family transcriptional co-repressors and WRKY transcription factors (TFs). In this project, we aim to investigate how two WRKY TFs work together with the TOPLESS-related 1 co-repressor (TPR1) during an nlp24-triggered immune response. You will test for their physical association and evaluate the contribution of the WRKY TFs to PAMP-triggered immunity outputs.

Learned skills: RNAseq, plant genotyping, cloning, generation of stable Arabidopsis transgenic lines, qRT-PCR, and investigation of protein-protein interactions via FRET and coimmunoprecipitation assays.

New hormonal regulators of plant recovery after an immune response

Project description: After pathogen recognition, plants initiate immune reactions, which lead to adverse effects at the level of plant growth and development. For sustainable crop production, plants are required not only to defend themselves but also to recover quickly, but the latter is poorly understood. This project aims to identify new regulators of plant recovery after an immune response. The project has two branches. In the first branch, we utilize the plasma membrane-bound receptor-like protein 23 (RLP23), which is triggered by a pathogen-associated molecular pattern (PAMP) peptide nlp24 and activates leaf senescence. We found that this process involves a WRKY transcription factor. This part of the project will test the hypothesis of whether and how the WRKY-regulated hormone signaling protects plants from an overstimulated immune system. In the second branch of the project, you will map a gene required for the viability of the plant shoot apical meristem during a salicylic acid (SA)-triggered immune response. We have identified a mutant that is unable to recover the meristem activity after the SA treatment. Bulk segregant analysis and sequencing pointed to several candidates. In this part of the project, you will finalize the identification of the underlying gene.

Learned skills: plant genotyping, cloning, generation of stable Arabidopsis transgenic lines, qRT-PCR, CRISPR/Cas9 mutagenesis, bulk segregant analysis, phenomics data collection and processing

Proteomics-based identification of the regulators of photosynthesis in stressed plants

Project description: Photosynthesis is a key process for producing plant biomass, but this process is sensitive to environmental perturbations including immune stresses. Here, we aim to find key players (proteins) that help stressed plants maintain their photosynthetic capacity during and after an immune response. These proteins could become promising targets for improving crop resilience.

The project has two directions. In the first, exploratory direction, you will use a novel method of targeted protein capture to find what proteins bind promoters of photosynthesis genes and regulate their expression during an immune response. The method combines the CRISPR-Cas9 technology with the proximity labeling of proteins via TurboID. In this system, called TurboID-dCas9, Cas9 is mutated to guide TurboID to the promoter of interest, but it does not cut DNA (dead Cas9 or dCas9). You will use available Arabidopsis thaliana plants expressing TurboID-dCas9, which was programmed to target one photosynthesis gene involved in light harvesting. You will apply the defense hormone salicylic acid to activate immunity and repress the expression of the photosynthesis gene. Proteins binding to the promoters of this gene will be labeled with biotin and further identified using proteomics methods in collaboration with the Biochemistry Laboratory at Wageningen University. We anticipate finding proteins that regulate photosynthesis at the level of DNA accessibility and transcription during immunity stress, which will form a basis for future functional analyses.

In the second, more targeted direction, you will create Arabidopsis plants with mutated candidate regulators of photosynthetic genes via easy-to-use and highly efficient CRISPR/Cas9 mutagenesis methods established in our lab. These mutants will be further tested for their photosynthetic performance and growth in the presence of salicylic acid.

Learned skills: processing of proteomics and other datasets in R/Python, CRISPR-Cas9 mutagenesis in plants, plant transformation, cloning, western blotting, protein affinity purification

Genome-wide association studies of immunity and growth traits in lettuce

Project description: Identification of new genes responsible for immunity and growth traits in lettuce is of critical importance for improving our understanding of lettuce physiology and improving it in the breeding process. In this project, you will perform a genome-wide association study (GWAS) of a trait or follow up on the leads from previous GWAS analyses conducted in our laboratory. We work with traits related to the pathogen recognition and growth control during and after an immune response. Project details depend on the trait that we are currently interested in.

Learned skills: GWAS, genome and synteny analyses, CRISPR-Cas9 mutagenesis in plants, cloning

Protein-protein interactions regulating growth-immunity trade-offs in Arabidopsis

Project description: Plants have evolved a sophisticated innate immunity that relies on phytohormones such as salicylic acid (SA) and jasmonic acid (JA). While these hormones play crucial roles in disease resistance, immune activation often comes at the cost of plant growth. This internship aims to test for the molecular interactions between NPR1, an SA receptor, and the BAP-D module proteins that allow integration of hormonal and light signals to drive growth by cell division and elongation under favourable conditions in Arabidopsis. The BAP-D module includes Phytochrome-Interacting Factors (PIFs), Auxin Response Factors (ARFs), and Brassinosteroid-responsive BZR1 and integrates hormonal and light signals to drive growth by cell division and elongation under favourable conditions. In this project, the student will focus on protein-based techniques to test for physical interactions between NPR1 and components of the BAP-D module. You will also search for new interactors in NPR1 in the context of growth-defense trade-off using proximity labelling. Core methodologies will include: molecular cloning, co-immunoprecipitation, and computational analysis of the proteomics data. The internship will provide hands-on experience with protein interaction techniques while contributing to ongoing research.

Learned skills: molecular cloning, co-immunoprecipitation, preparation of samples for proteomics analysis, and analysis of the proteomics data.