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In this project the genome of a novel yeast species associated with pest insect Nezara viridula will be sequenced and investigated.

The main aim is to get a better understanding of why this yeast is associated with the insect, if it produces vitamins and other useful molecules for the insect. A pure culture is available from which high-quality DNA will be isolated and sent for long read (e.g. PacBio) sequencing. When the genome is assembled and annotated hypotheses can be formulated for further research. Beside the major part of this research project there is room for additional experiments to solve related research questions.

A second question is the location of the yeast. It is a 20-um long rod-shaped microbe, which can be isolated from the saliva and salivary glands of the insect. Verifying where it is exactly located will make it easier to determine its potential role. Therefore, an effort will be made to locate the yeast with microscopy and molecular techniques (e.g. fluorescent in situ hybridization) on dissected insect tissues.

Another question we have is about potential effector proteins produced by other insect-associated microbes. The genomes of several other N. viridula-associated microbes are available from prior research in which we showed that these microbes can suppress plant defenses. How this is achieved is unclear. Searching for potential effector proteins in their genomes will hopefully identify targets for future research. We for example know that one of the microbes has genes required for a type III secretion system involved in transfer of effectors. Whether these effectors are produced and transferred to the insect host or to the plant, from which the insects feed, is unclear.

Daily supervisor

Silvia Coolen

From prior research we noticed that microbes associated with pest insect Nezara viridula can potentially influence insect preference for host plants. Preliminary experiments showed that insects preferred to lay eggs on plants inoculated with their own associated microbes. We also know from prior research that these microbes can suppress plant defenses, likely benefitting the offspring of N. viridula. To confirm our hypothesis a stable rearing population will be maintained and used for choice assays in which plants are inoculated with diverse insect-associated microbes. If insect-associated microbes alter host plant choice, these findings may be of direct use in greenhouse systems where management of N. viridula is a struggle. Making trap crops that are inoculated with these microbes may lure insects to one greenhouse area where they can be manually removed. In addition, an attempt will be made in sterilizing insect eggs to generate microbe free animals that can be reinoculated with specific microbes to study their separate functions.

Techniques that will be used are predominantly microscopy, dissection, microbiology techniques, insect rearing and field- work (e.g. collecting insects). If microbe free insects are generated additional experiments can be started to reintroduce microbes and determine their function (e.g. detoxification of plant secondary metabolites).

As a side project we would also like to attempt to isolate Commensalibacter, which we could not isolate from our insects yet, although we have some clues of how to do it now. The function of this microbe is still unclear. Possibly it is involved in protecting the insect against pathogens.

Daily supervisor

Silvia Coolen

This project is part of the Fruit of Knowledge project, currently running at UU and WUR, that focuses on cultured fruit as a sustainable technology for industrial fruit production. A 4-step process will provide proof-of-concept for this technology: 1) plant meristems are multiplied 2) flowering is induced in these meristems 3) fruit-set is induced using phytohormones and 4) fruitlets are grown in a bioreactor. Aim of the UU-based part of the project is to sustain meristem cultures and to be able to induce direct flowering in these meristems, resulting in the formation of single flowers without the development of vegetative tissues.

Recent developments in CRISP/Cas9 technology have led to the creation of dCas9 activation methods. Here, Cas9 has two missense mutations that shut down the nuclease activity and make it a dead Cas9 (dCas9). This dCas9 protein is fused to an activator complex that can activate transcription by binding on promotor sequences of target genes. We aim to use this Crispr-activation (CRISPRa) system, both to induce meristem formation as well as to transition these meristems into flowers. The past year, we have developed this technique for tomato and tested it for selected targets in protoplasts. Next step will be testing this system in planta.

This MSc internship project focuses on the application of this technique in planta to induce ectopic shoot meristem formation. When successful, floral transition and/or floral identity genes will be activated in these meristems to induce direct flowering, using the CRISPRa system.

During this internship you will learn a scala of bio-molecular and plant physiological skills that include: MoClo cloning, CRISPR pipeline and guide design, plant transformation, confocal/fluorescence microscopy, photography, tissue culture and general lab skills. 

Daily supervisor

Niels Peeters (n.r.peeters@uu.nl);

Heterochronic bolting corresponds to a sustained rapid elongation of the primary stem in rosette plants while still in the vegetative growth phase. Heterochronic bolting is a major problem in the cultivation of lettuce (Lactuca sativa L.), especially when grown in high densities in hydroponics, as in the case of teenleaf lettuce. Heterochronic bolting, or leggy lettuce, results in less leaf biomass and poor head development, both of which are essential for its marketability. Development of lettuce varieties with enhanced heterochronic bolting resistance is thus of great economic importance. As part of LettuceKnow (), a research program aiming to establish lettuce as a model crop for research, we aim to identify lettuce genes/gene variants mediating environment-controlled stem development.

We recently performed a large GWAS screen, using 200 L. sativa cultivars, that investigated the combined effects of light intensity and temperature on heterochronic bolting.

The proposed MSc project focuses on obtaining a more detailed molecular basis of vegetative internode elongation. We have a F2 generation of a cross between an accession that shows major elongation under inductive conditions (low light and high temperature) and an accession that shows minor elongation. This F2 generation can be utilized for Bulked Segregant Analysis (BSA), to identify the molecular basis of this phenotypical difference between accessions.  In addition, genotyping and phenotyping of already available gene-edited CRISPR/Cas9 lines (T1 and T2 generation) for a priori selected candidate genes will be part of the project.

Photosynthesis is a key process for producing plant biomass, but this process is sensitive to environmental perturbations. 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 or a pathogen-associated molecular pattern (PAMP) 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 ľ¹Ï¸£ÀûÓ°ÊÓ. 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 tested further for photosynthetic performance and growth in the presence of salicylic acid or PAMP.

Learned skills

Processing of proteomics and other datasets in R/Python, CRISPR-Cas9 mutagenesis in plants, plant transformation, cloning, Western blotting.

Supervisors

Dmitry Lapin (d.lapin@uu.nl)

Diseases of plants in nature and agriculture are unavoidable. However, even successful defense responses are associated with defects in plant growth and development. In medical research, it became evident that timely inactivation of the immune system is essential for health (e.g., ‘long COVID-SARS’ syndrome). However, little is known about how plants switch off their immune system and return to a normal physiological state. This knowledge would be critical to developing crops that are resistant to the pathogen insult and quickly recover from the infection. In this project, you will search for regulators of plant recovery from immunity stresses.

You will use an available collection of ~40 Arabidopsis thaliana mutants for transcription factors (TF) and selected metabolic and growth regulatory pathways. The collection also contains mutants with poor recovery after an immune reaction as positive controls, already established but not yet well characterized in our lab. First, you will screen these mutants for their ability to activate the immune system in response to pathogen-associated molecular patterns (PAMPs). For this, you will measure reactive species oxygen (ROS) burst and, for selected mutants, defense gene induction after the PAMP application. Second, you will trace the growth rates of these mutants after the PAMP application or infections with the downy mildew pathogen using the standalone phenotyping system FluorCam. Finally, based on the obtained data, you will propose hypotheses about involved molecular pathways and initiate testing one prospective hypothesis with appropriate methods.

Learned skills

ROS burst assays, infection assays, Western blotting, RT-qPCR, phenotyping using plant imaging systems, and modeling of growth rates in the framework of mixed statistical models.

Supervisors

Dmitry Lapin (d.lapin@uu.nl), Daan Weits (d.a.weits@uu.nl) and Iñigo Bañales (i.banalesbelaunde@uu.nl)

Current efforts to improve crop disease resilience focus on finding new immune receptors and changing the defense hormone balance. However, avoiding uncontrolled immune activation is as important for plant health. It is unclear how plants switch off their immune responses and return to a normal physiological state. In this project, you will find novel candidate regulators of plant recovery from an immune response using Arabidopsis thaliana as a model system.

In the consortium LettuceKnow, we traced immunity-associated growth and recovery in ~150 lettuce varieties using high-throughput phenomics technologies, which allows us to identify candidate regulators of these processes. However, the list of candidates is still too long. Your project thus serves as an orthogonal, high-resolution approach to speed up translational work in lettuce.

You will use a collection of ~600 sequenced Arabidopsis mutants (HEMs) already available in our group. You will screen these lines in a high throughput format to determine their ability to grow and recover from immune reactions. These immune reactions will be triggered by the defense hormones salicylic acid (SA) and jasmonic acid (JA), the pathogen-associated molecular pattern (PAMP) nlp24, and the downy mildew pathogen. You will measure the rosette size as a proxy of plant health using standalone phenotyping systems or . Since all mutants have been sequenced, you can connect the growth and recovery potentials to a few candidate genes using statistical modelling approaches established in the lab.

Learned skills

Semi-automated plant phenotyping, statistical modelling, data processing in R/Python.

Supervisors

Dmitry Lapin (d.lapin@uu.nl) and Bart Schimmel (b.c.j.schimmel@uu.nl)

During this MSc. project you will develop a novel tool for precise detection and analysis of DNA methylation. DNA methylation plays a crucial role in gene regulation and cellular processes, yet current methods for its detection can be costly, time-consuming, or lack specificity.

Project Goal

You will engineer a new protein tool by fusing methylation-binding domains (MBD) to the FokI DNA nuclease. This engineered protein will selectively cleave methylated DNA sequences while leaving unmethylated DNA intact.  You will grow and purify this protein to test the optimal conditions for use by digesting genomic DNA and performing qPCR over sites with known DNA methylation status.

Key Aspects of this Project

• Design and Construction: Using molecular cloning techniques, you'll design and assemble the genetic constructs encoding your novel fusion protein.
• Protein Expression and Purification: Express and purify your engineered protein to be used in in-vitro assays.
• Functional Validation: Test the specificity and efficiency of your methylation detection tool.

Skills You'll Gain

• Hands-on experience in synthetic biology and protein engineering.
• Expertise in molecular cloning techniques (PCR, restriction enzyme digestion, ligation).
• Experience in in-vitro assay development
• Understanding of DNA methylation and its biological significance.

Further Reading

Origins of Programmable Nucleases for Genome Engineering. Chandrasegaran and Carroll. JMB 2016. Designed nucleases for targeted genome editing. Lee et al., New Phytologist 2015. DNA Methylation Readers in Plants. Grimanelli and Ingouff. JMB 2020.

Daily supervisor

Dr. Jason Gardiner (j.l.gardiner@uu.nl)