Education
Educational projects
Themes of the PMI group
- Beneficial microbes and plant immunity - Corné Pieterse
- Regulation of plant immunity in leaves and roots - Saskia Van Wees
- Microbial (meta)genomics of plant-associated microbes - Ronnie de Jonge
- Recruitment of beneficial microbiomes - Roeland Berendsen
- Plant ImmunoMetabolism- Arsheed Sheikh
When you are interested in performing a MSc student research project in our group but you do not yet know exactly what you want, please contact Corné Pieterse (C.M.J.Pieterse@uu.nl; if you want to orient yourself on which topic would suit you best), or one the other staff members mentioned above. Below a number of concrete examples of projects that can be done in the PMI group (but this is not exhaustive).
Host microbiome research has emerged as a new imperative in the life sciences. The realization that thousands of microbial species are an integral part of the biology of eukaryotic hosts and expand their genomic potential comes with great new opportunities for, for example, future microbiome-assisted agriculture. Recent discoveries that root-associated microbes support plant growth, nutrition, and immunity fostered bio-based crop system approaches to reduce the use of agrochemicals. However, the dream of microbiome-assisted agriculture as a genuine sustainable contribution to global food security will require a deep understanding of the genetic, ecological and evolutionary principles underlying plant-microbiome interactions. Students are very much welcome to contribute to our endeavour of doing science at the forefront, while contributing meaningful knowledge to our society of the future. There are several opportunities for performing a research project under supervision of one of our scientists. Below a number of examples, although this is not exhaustive.
If you are interested in the general topic but do not see an immediate topic that you like best, feel free to first contact Corné Pieterse (C.M.J.Pieterse@uu.nl), who can help guide you through the current options.
The plant immune system not only fights pathogens but also functions as a gatekeeper that favours life-supporting microbes to colonize on the host, leading to benefits of the plants, such as growth promotion, enhanced nutrient uptake, and enhanced biotic and abiotic stress resilience. We discovered that coumarins, which are fluorescent secondary metabolites that are secreted by plant roots, play a very important role in the communication between plant roots and beneficial members of the root microbiome, such as our model plant growth- and health-stimulating Pseudomonas WCS417 strain (Stassen et al., 2021). On the one hand coumarins have a selective antibiotic activity and therefore play an important role in selecting which microbes can grow on the root surface and which ones not (Stringlis et al., 2018). On the other hand, coumarins seem to be important signal molecules that travel from the root to the shoot, where they play a role in priming the immune system and protecting the plant against pathogen attack. Some of the research questions that we are currently addressing (and in which you can participate) are:
- Do coumarins also function as mobile signals that travel from the roots to the shoots to prime the plant's immune system?
- What makes a rhizobacterium sensitive or insensitive to coumarins that are secreted by plant roots, and make that they can grow or not on the root surface?
- What is the link between coumarins and their capacity to mobilize iron from the soil? And how does this interact with their selective role in root microbiome assembly?
Our research on bidirectional signalling along the microbiome-root-shoot axis provides a firm knowledge basis to harness the power of the plant’s second genome for future microbiome-assisted crop systems. Your help during a 6-9 months research internship is very much welcome! .
References
- Stassen, M.J.J., Hsu, S.-H., Pieterse, C.M.J. and Stringlis, I.A. (2021). Coumarin communication along the microbiome-root-shoot axis. Trends in Plant Science 26: 169-183.
- Stringlis, I.A., Yu, K., Feussner, K., De Jonge, R., Van Bentum, S., Van Verk, M.C., Berendsen, R.L., Bakker, P.A.H.M., Feussner, I. and Pieterse, C.M.J. (2018). MYB72-dependent coumarin exudation shapes root microbiome assembly to promote plant health. PNAS 115: 5213-5222. .
Techniques
n vitro plate assays – plant disease bioassays – plant and bacterial mutant analyses – gene expression analyses – bacterial growth assays – coumarin assays – NPEC root colonization
Daily supervisor
Max Stassen (M.J.J.Stassen@uu.nl)
Induced systemic resistance (ISR) in plants is triggered by beneficial rhizobacteria such as Pseudomonas simiae WCS417. It starts with successful root colonization by WCS417 and then systemic defense responses are observed in the leaves following attack by pathogens. We previously found that WCS417 can trigger coumarin biosynthesis in the roots of Arabidopsis and the key components of ISR have roles in the accumulation and processing of coumarins before their release in the rhizosphere. There is also evidence that coumarin biosynthesis in Arabidopsis might be critical for root-shoot communication during ISR establishment. An ortholog analysis of coumarin biosynthesis genes between A. thaliana and 54 sequenced plant species revealed that the coumarin biosynthesis pathway widely exists in dicots plant species like Cicer arietinum (chickpea), Malus pumila (apple), Solanum tuberosum (potato), Solanum lycopersicum (tomato) and Glycine max (soybean). In this regard, understanding the mechanisms underlying bacterial-induced plant defense response will facilitate the development of better and novel strategies for disease control and sustainable crop production practices.
Aim of this project
The current project aims to conduct an in-depth investigation into the significance of coumarins in plant resistance. Induced systemic resistance assays (in vivo/vitro) will be used to confirm the WCS417-mediated effect on plant defense system across various coumarin mutants. Next, we will quantify the defense priming response within the leaf in response to inoculation of WCS417 among mutants. Our objective is to identify the pivotal components responsible for the activation of the WCS417-mediated plant defense pathway. Finally, the expression patterns of coumarin biosynthesis genes will be analyzed with qRT-PCR to understand how the root-inoculated WCS417 reprograms coumarin biosynthesis and orchestrate the coumarin reallocation among different tissues.
The techniques that will be used:
- Arabidopsis In vitro/vivo bioassays
- Microbial growth, root inoculation, and evaluation of microbial colonization.
- Induced systemic resistance (ISR) in vitro/vivo bioassays.
- Gene expression profiling via qRT-PCR.
- In vitro defense hormone priming assay.
Daily supervisor
Dr. Shuhua Hsu (s.hsu1@uu.nl)
 and field trial in Nicaragua (Right).jpg )
Figure: Cute coffee seedlings in NPEC (Left) and field trial in Nicaragua (Right).
Are you passionate about the fascinating world of agriculture and intrigued by the mysteries that lie beneath the soil? If so, we have an exciting research opportunity for you! We are seeking enthusiastic master's students to join us on a journey into the realm of coffee production. Our research project aims to unravel the intricate relationship between this beloved crop and the microorganisms that inhabit its roots. In this project, we will decode the taxonomic and functional composition of the root microbiome in coffee and cocoa genotypes grown in diverse geographic locations and under varying cultivation conditions. By examining the hidden microbial communities present in the soil surrounding these plants, we aim to shed light on their role in enhancing crop resilience. As a master's student, you will:
- Conduct greenhouse experiments with coffee plants (is it cool!) and collect soil samples.
- Interact with researchers, students, and farmers from different countries across Africa, Asia and Europe.
- Use advanced molecular and sequencing techniques to uncover the diverse microorganisms living in the roots of coffee and cocoa plants.
- Learn bioinformatics skills to explore the relationships between microorganisms and plants to understand their contributions to crop well-being.
- Drive sustainable practices: Your research findings will inform sustainable agriculture practices, promoting productivity while reducing chemical inputs.
Further reading
This project is part of the EU-project Breeding for coffee and cocoa root resilience in low input farming systems based on improved rootstocks ().
Daily supervisor
Yang Song (y.song1@uu.nl)
Drought, increasingly intensified by climate change, poses a significant threat to global food security. Plant-associated microbiomes are crucial in enhancing plant resilience to drought. However, traditional methods for identifying beneficial microbes are time-consuming and labor-intensive. You will work on developing a novel strategy that leverages machine learning (ML), metagenomic sequencing and high-throughput phenotyping to efficiently predict and select optimal microbiome combinations for drought resistance. This approach will accelerate the high-throughput identification of beneficial microbes based on their functional profiles, addressing the urgent need for microbe-assisted agriculture.
Different SynComs combinations will be designed based on the functional profile of each strain in the Arabidopsis-associated microbial collection in our lab. You will evaluate the effect of each SynCom on Arobidopsis root architecture and drought resistance with the support of the Netherlands Plant Phenotyping Centre (NPEC) facilities. ML prediction models of plant drought resistance base on the phylogenetic and functional information of these 200 different SynComs combinations will be generated. The Top10 best predictors (strain) of plant drought resistance from the ML model will be extracted and the causal relationships between individual predictor and plant drought resistance will be tested by conducting bioassays in soil and in vitro systems at NPEC.
Further reading
Our recent research has demonstrated that plant microbiomes hold significant predictive power for plant performance. Moreover, with ML prediction models, it is possible to identify the microbial predictors of plant performance.
Daily supervisor
Yang Song (y.song1@uu.nl)& Yizhu Qiao (y.qiao1@uu.nl)
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The region of soil surrounding plant roots, the rhizosphere, is rich with microorganisms that can form close associations with plants. The plant growth-promoting bacterium Pseudomonas simiae WCS417 is known to colonize the roots of the plant model organism Arabidopsis thaliana. This association leads to changes in the morphology of the plant, such as an enhanced shoot growth and changes in the root system architecture. Additionally, it can also enhance plant health, by making it more resilient against plant pathogens and environmental stress. This project aims to further our understanding in the response of Arabidopsis thaliana to plant growth-promoting WCS417 rhizobacteria, in the context of biotic and abiotic stresses resilience.
We capture these plant responses using high-throughput, image-based phenotyping techniques in a new facility on the campus of Utrecht ľ¹Ï¸£ÀûÓ°ÊÓ: The Netherlands Plant Eco-phenotyping Centre (NPEC). These image-based phenotypic techniques are a non-destructive and time-saving way to generate daily phenotypic data, significantly enhancing our understanding of plant-microbe interactions.
As a master student in this project, you will:
- Design and conduct Arabidopsis bioassays incorporating biotic or abiotic stress factors
- Learn how to grow and apply microorganisms to plant roots
- Get the opportunity to work in a state-of-the-art imaging technology platform
- Develop bioinformatic skills by analyzing large phenotypic datasets
Plant phenotyping – NPEC – Beneficial microbes – Data analysis
Daily supervisor
Sophie Vijverberg (s.g.f.vijverberg@uu.nl) and Valerian Meline
Plant hormones play a vital role in the regulation of plant immunity. In recent years, our lab studied the gene regulatory networks (GRNs) activated by some of these hormones based on high-density transcriptome time series data of plants treated with the defense-related hormones salicylic acid (SA), jasmonic acid (JA) and abscisic acid (ABA). We revealed some of the architecture and dynamics of the GRNs activated by these hormones. Additionally, because we also performed combined hormone treatments, we were able to study how the different networks interact with each other. Such (complex) interactions between hormone networks are known as crosstalk. Although we did a basic analysis on crosstalk based on our data, much is still to be discovered. For example, while we predicted potential key crosstalk regulators using a bioinformatics approach, we did not thoroughly test their role in the lab yet. Also, the generation of new, publicly available datasets generated by other scientists could allow new bioinformatics approaches to better predict key crosstalk regulators. In the proposed MSc project some of these open ends can be addressed. For example, hormone treatment assays and pathogen/insect bioassays with mutants of predicted crosstalk regulators can be performed to verify their importance within hormone GRNs for plant immunity. Also, molecular assays such as western blots or interaction assays can be done to elucidate how certain key transcription factors that act in one hormone pathway are affected by another hormone. Alternatively, a yeast-one hybrid screen may be done to find all transcriptional regulators of key genes in the different hormone GRNs. Finally, different bioinformatic analyses to, e.g., predict novel regulators of crosstalk are also possible. Because this is not all possible in a single project, the project can be adjusted to the wishes of the student.
Hormone treatment / disease bioassays with transcription factor mutants ∙ Western blots ∙ Yest one-hybrid ∙ Yeast two-hybrid ∙ qPCR ∙ Bioinformatics
Daily supervisor
Niels Aerts (n.aerts@uu.nl)
Although many scientific studies have aimed at unravelling the plant defense system, one important regulatory level is continuously overlooked: post-transcriptional regulation. Understanding this level of gene expression regulation is essential since the transcript levels in a cell do not necessarily reflect what is translated into proteins. Several post-transcriptional regulatory mechanisms control the fate of mRNA molecules, including the nuclear retention of mRNAs, the translocation of mRNAs to processing bodies & stress granules, and the engagement of ribosomes with mRNAs. We aim to elucidate how such post-transcriptional regulatory mechanisms contribute to plant immunity.
The MSc project will focus on nuclear mRNA retention. This regulatory mechanism can prevent premature release of incompletely spliced pre-mRNAs to the cytoplasm. Additionally, by retaining specific mRNAs in the nucleus, while releasing others to the cytoplasm, a plant can control which mRNAs are translated into proteins in response to diverse stimuli. This might function in saving energy during stress conditions and quickly recovering once the stressor disappears. The project consists of (a combination of) two subprojects: (1) Identify nuclear-retained transcripts: Identify RNA retained in the nucleus during activation of plant defense by comparing the nuclear RNA pool to the total RNA pool. For this, an RNA-seq experiment has already been carried out. The MSc project will aim to verify these findings with single gene expression analyses and/or microscopic visualization of selected transcripts carrying a fluorescent tag. (2) Test putative regulators of nuclear retention: Elucidate the role of predicted regulatory proteins (e.g., RNA binding proteins, nuclear pore complex) in nuclear mRNA retention & immunity. Together, these subprojects encompass various molecular techniques, which can be complemented by phenotypic disease assays.
Molecular biology ∙ Plant immunity ∙ Post-transcriptional regulation ∙ Nuclei isolation ∙ RNA localization & qPCR ∙ Cloning ∙ Confocal microscopy ∙ Disease bioassays
Daily supervisor
Tessa Visscher  (t.w.visscher@uu.nl)
Plant diseases caused by pests and pathogens represent a major constraint for agriculture worldwide. These problems are aggravated by a changing climate, particularly harsh conditions such as drought, flooding, and raising temperatures. These abiotic factors negatively impact plant performance not only as single stress but also influence plant immunity levels thus potentially making the plants more vulnerable to pathogens and insects. Plant performance under various stress conditions relies on the activation and repression of different hormone-inducible responses. The role of individual hormone pathways in controlling plant resistance to single biotic and abiotic stresses is well known. However, the interaction between these hormone networks when plants experience multiple stresses at the same time, a situation that occurs frequently in nature and agriculture, has received little attention.
This MSc project aims to contribute to the investigation of the effect of elevated temperature on immune responses triggered by pathogens in plants. The student will be involved in the elucidation of which sectors within the plant hormone gene regulatory networks are differentially engaged under dual stress conditions.
Therefore, Arabidopsis thaliana plants will be cultivated under a range of temperature conditions, and simultaneously infected by a pathogen. Expression levels of stress-responsive marker genes will be analysed to correlate phenotypic changes with molecular alterations, potentially identifying key genes that could enhance plant resilience through early signalling mechanisms. During this MSc project you will run bioassays with different pathogens, carry out plant hormone treatments, perform qPCRs for gene expression analysis and work with mutant lines affected in hormone and temperature signalling in order to decipher the key regulators involved in the immunity-high temperature interaction.
Plant pathology/physiology ∙ Molecular biology · Hormone treatment ∙ Disease bioassays ∙ Gene expression profiling via qPCR
Daily supervisor
Julia Ruiz Capella (j.ruizcapella@uu.nl)
The global average temperature continues to increase yearly, and 2023 is known as the warmest year ever recorded. In connection with this phenomenon, the number of heat wave events has also increased and spread across various regions throughout the world.
While plants must protect themselves from heat stress, they also have to deal with other threats like pathogen infection. Preliminary experiments showed that heat waves (33°C) increase Arabidopsis susceptibility towards grey mould (Botrytis cinerea). On the other hand, beneficial microbes have proven to help plants ameliorate abiotic stress effects and enhance their resistance to pathogens in single stress experiments. This project aims to investigate the potential of beneficial microbes to enhance plant resistance against the combined stress of heat and pathogen infection. Therefore, we will grow Arabidopsis in soil mixed with different beneficial microbes from the PMI collection. We will subject them to a heat wave (33°C) and after a short period of recovery, we will infect the plants with pathogens to observe the impact of priming and heat treatment on the development of disease. Molecular analyses will provide insight into the underlying mechanisms, for which hyperspectral images will be generated in the NPEC facility and gene expression analyses by qPCR will be performed. Arabidopsis mutants with a defect in priming, defense signaling or heat stress responsiveness will be assayed to verify the role of molecular players in the interaction.
Plant disease assays ∙ Combined stress ∙ ISR ∙ Systemic signaling ∙ Image analysis ∙ Gene expression profiling via qPCR
Daily supervisor
Rahmi Masita (r.masita@uu.nl)
No specific projects for this academic year, but feel free to contact Ronnie for further inquiries when you are specifically interested in Microbial (meta)genomics of plant-associated microbes or the use of AI-technology in plant-microbe interactions research (R.deJonge@uu.nl).
Potato is one of the most important food crops globally but conventional potato cultivation relies heavily on agrochemical inputs, raising environmental and health concerns. However previous studies have unveiled the significant influence of microbial communities on plant health and growth. The microbiome, composed of various microbes associated with plants, contributes to nutrient acquisition and protection against pathogens and stresses. Our research has shown that the microbiome composition of potato tubers can provide valuable insights into the health of the emerging potato plant. Moreover, we are able to predict the health of the potato plant by sequencing the microbiome of the tubers.
In this new project we aim to understand how the plant microbiome benefits potato and how we can use this in sustainable agriculture. As a student involved in the project, you will isolate and characterize microbes from potato rhizospheres and soils. You will study how particular microbes in isolation or in communities interact with the host plant and how they ultimately affect plant health. By participating in this project, you will gain valuable insights into the complex interactions between plants and microbes, enhance your bioinformatic and analytical skills, and contribute to the development of sustainable potato cultivation practices.
Daily supervisor
Juan Sánchez ( j.j.sanchezgil@uu.nl)
Improving agricultural resilience to drought stress is essential for sustainable food production, especially as climate change intensifies environmental challenges. One promising approach is harnessing the power of the root microbiome to enhance plant growth and stress tolerance. In this project, we aim to optimize potato breeding by identifying genetic traits that improve cooperation between potato plants and beneficial soil microbiomes. At the NPEC facility, using the HADES module, we will grow a diverse set of potato genotypes in plate assays. These assays will be inoculated with various soil microbiomes and individual bacterial strains to investigate their effects on root architecture and drought resilience. The ultimate aim of the project is to identify M-genes, plant genes involved in the enrichment of beneficial microbes in the microbiome.
As a master student, you will have the opportunity to:
- Design and conduct plate-based assays with different potato genotypes and microbiome inoculations
- Utilize advanced phenotyping technology to study root structures under drought stress conditions
- Analyze genetic and phenotypic data to identify traits that enhance beneficial plant-microbiome interactions
- Develop bioinformatics skills for large-scale data analysis
- This project offers a hands-on experience in cutting-edge plant science research, combining genetics and microbiome studies to develop more resilient crop varieties for future agricultural needs.
Daily supervisor
Jochem Huijben (j.huijben@uu.nl)
Plants under siege can recruit protective microbes to help defend against pathogens, but the mechanisms for this are often unknown. The model plant Arabidopsis thaliana has been shown to recruit protective Hpa-associated microbiota (HAM) upon aboveground infection of the obligate biotrophic oomycete pathogen Hpa (downy mildew), as part of the soil-borne legacy of inter-generational disease resistance. The Xanthomonas strain WCS2014-23 is a key member of the HAM and increases in phyllosphere abundance up to 16x upon Hpa infection while contributing to reduced disease severity. We previously carried out a large transposon insertion mutant screen (INSeq) on Xanthomonas sp. WCS2014-23 and identified several candidate genes potentially important for its recruitment. This project aims to establish a system for targeted mutagenesis in Xanthomonas sp. WCS2014-23 and carry out in planta competitive recruitment assays with fluorescently labelled strains to discover novel phenotypes predicted by the INSeq screen.
As a masters student in this project, you will:
- Analyze and interpret bioinformatic data from large transposon mutant screens
- Design and pursue a targeted mutagenesis strategy in Xanthomonas
- Learn advanced techniques for molecular cloning and transformation
- Work with plants, bacteria and oomycetes in complex interaction
Bacterial genetics – Molecular cloning – Beneficial microbes
Further reading:
Daily supervisor
Brandon Ford (b.l.ford@uu.nl)
Hyaloperonospora arabidopsidis (Hpa) is an obligately biotrophic downy mildew that is routinely cultured on Arabidopsis thaliana leaves that harbour complex microbiomes. We found that the culturing procedure proliferates Hpa-associated microbiota (HAM) in addition to the pathogen and exploited this model system to investigate which microorganisms consistently associate with Hpa. Pathogen-infected plants can selectively recruit HAM to both their roots and shoots and form a soil-borne infection-associated microbiome that helps resist the pathogen (Goossen et al (2023) Nature microbiology). You will work on identifing the function of HAM and expaining the underlying mechanism of HAM enrichment in leaves using metatranscriptome and genome analysis.
As a master student in this project, you will:
- Design and conduct sterile Arabidopsis bioassays responding to Hpa infection and bacteria inoculation
- Learn advanced techniques for microbiology, molecular cloning and transformation
- Work with plants, bacteria and oomycetes in complex interaction
- Develop basic bioinformatic skills
Daily supervisor
Yadong Shao (y.shao1@uu.nl)
With a growing population and a deteriorating climate change, we need to ensure food security while transitioning to more sustainable and efficient agricultural practices. Promoting soybean cultivation is essential, as it is a protein-rich crop that can support a shift from unsustainable animal-based diets towards a sustainable plant-based food system. To that end, microbiome-assisted agriculture has a key role in enhancing soybean development without relying on chemical inputs.
This project focuses on explore how different microbiomes affect soybean growth. Ultimately we want to identify beneficial microbes that can enhance plant development and drought tolerance. We have previously collected a diverse set of microbiomes from 100 different fields across Europe, which will be inoculated onto a selected soybean variety. The plants will be grown in the Helios system at the NPEC, a high-throughput phenotyping facility. The advanced measuring equipment will allow us to monitor even subtle differences in plant growth in response to different microbial communities. After the growth period, we will collect root samples and perform DNA extractions to analyze the microbiomes associated with each soil sample. Using advanced microbiome analysis techniques, we aim to identify microbes that promote growth and resilience in soybeans.
As a master student, you will:
- Learn about microbial inoculation techniques and apply them to inoculate soybean plants with diverse microbial communities.
- Collect data from advanced high-throughput phenotyping equipment and use it to evaluate differences in the growth of soybean plants in response to different microbiome treatments.
- Familiarise with lab techniques such as DNA extraction and sample preparation for microbiome sequencing to study plant-microbe interactions.
- Develop basic skills in bioinformatics and data analysis to identify beneficial microbes associated with enhanced soybean growth and drought tolerance.
- Gain overall experience in experimental design, data interpretation, and scientific thinking.
Daily supervisor
Maria Papadopoulou (m.papadopoulou@uu.nl)
Sulfur is the fourth essential plant nutrient, critical for many growth functions including enzyme activity, protein and oil synthesis and is needed for optimal photosynthesis, chlorophyll formation, and nitrogen fixation. Given the decline in soil sulfur in recent decades, there is a necessity for sulfur fertilization in crops and grasses, with crops like Brassica napus (oilseed rape) particularly susceptible to sulfur deficiency. The sulfur-containing metabolic augmentation provided by beneficial microbes to host plants could be harnessed as an efficient and sustainable biofertilization strategy.
In this project, we aim to screen the microbiome associated with Arabidopsis (same family as oilseed rape) for their sulfur augmentation properties. We want to test bacterial synthetic communities (SynComs) and their individual members from the collection of the Plant-Microbe Interactions (PMI) group for their potential to enhance Arabidopsis growth under sulfur deficient conditions. The positive hits will be tested on Brassica napus plants for their potential on crop plants. Finally, the impact on microbial sulfur metabolism, including the regulation of sulfur regulons, will be examined to decipher the molecular mechanisms by which sulfur compounds are produced by microbes to be utilized by host plants. The student working on this project will first optimize the sulfur deficient media conditions for both Arabidopsis and Brassica napus in the lab. qPCR of sulfur-deficiency marker genes will be conducted to verify low-sulfur responsiveness of the plants. Subsequently, the beneficial effect of SynCom members will be tested by comparing the fresh weight and phenotype of colonized to non-colonized plants. Colony forming unit (CFU) assays will be used to test the relative interaction affinity of different microbes with Arabidopsis during S- conditions. Sulfur cues are sensed by TOR (Target of Rapamycin), which is a central signaling pathway regulating growth and nutrient signals in plants (Sheikh et al, unpublished). Therefore, the boost of host sulfur machinery by the microbes will be tested by activation of TOR using Western blotting. Finally, transcriptome changes in microbial sulfur metabolism, including the components of the sulfur regulon, will be analyzed to identify the potential sulfur compounds produced by the microbes and utilized by plants during sulfur deprivation.
The student will learn
Microbial growth and colonization assays, plant phenotyping and fresh weight assays, qPCR for quantitative transcriptomics, western blotting for protein related work
Molecular biology ∙ Plant Nutrition ∙ Sulfur metabolism ∙ qPCR ∙ Western Blotting
Daily supervisor
Arsheed H. Sheikh (a.h.sheikh@uu.nl)
RNA modifications are crucial post-transcriptional regulatory steps that determine RNA fate. Among these modifications, the methylation of adenosine residues, known as m6A, is particularly prominent. While the role of m6A is well-characterized in animal systems, its function in plants has only recently become a focus of active research. In plants, m6A modifications regulate growth, development, and responses to abiotic stresses. In addition, our recent research has demonstrated a role of m6A in plant immunity, showing a decrease in global m6A levels when plants are treated with the bacterial flagellin (flg22) peptide, which triggers innate immunity called pattern-triggered immunity (PTI)1.It is known that flg22 also induces changes in chromatin states, priming plants for enhanced defense responses to subsequent pathogen infections2. Moreover, in mouse embryonic stem cells m6A has been linked to chromatin remodeling, primarily through its impact on chromatin-associated RNAs (carRNAs)3. We therefore hypothesize that m6A-modified RNA contributes to PTI-induced plant immune priming, particularly through its effect on chromatin states.
In this project, we aim to understand the role of m6A in orchestrating plant defense priming during PAMP treatments. Our goal is to investigate whether m6A methylation in chromatin-associated regulatory RNAs (carRNAs) impact plant immunity responses. The primary objective will be to assess how bacterial flg22 and fungal chitin influence m6A methylation levels in carRNAs. Subsequently, the effect of carRNA modifications on chromatin accessibility will be examined using a DNase I digestion assay2
To determine the methylation levels of carRNAs after PAMP treatments, chromatin will be extracted from Col-0 Arabidopsis plants, followed by RNA extraction as described earlier 4. The m6A status of the extracted RNA will be assessed using dot-blot analysis with an m6A-specific antibody. Experimental controls will include m6A machinery mutants such as mta, fip37, vir1, and the 35S:ALKH10B overexpressor, which is an eraser of m6A. Chromatin accessibility will be evaluated by DNase I digestion of chromatin from both mock-treated and PAMP-treated plants. In addition to wild-type Col-0 plants, hypomethylated m6A mutants will be included to ensure rigor and reproducibility in the results.
You will learn
Plant Growth in specialized media and stress conditions. Treatment of plants with different PAMP elicitors. Chromatin isolation and carRNA isolation. Dot blot and RNA work. DNase1 digestion of chromatin.
Literature
- Prall and Sheikh et al., The Plant Cell 2023
- Sheikh et al., Nucleic Acids Research 2023
- Liu et al., Science 2021
- Conrad and Orom Methods Mol Biol 2017
Molecular biology ∙ Plant Immunity ∙ RNA modification and Epi-transcriptome ∙ qPCR ∙ Disease bioassays ∙ Western Blotting
Daily supervisor
Arsheed H. Sheikh (a.h.sheikh@uu.nl)