2015-­‐2016 Internship descriptions Track coordinator: Robert Schuurink Identification of Thrips Effector Proteins and their in planta Targets
Thrips (Frankliniella occidentalis) is a major plant pest that imposes a substantial pressure
on crop cultivations. It has a wide host range, which includes cut flowers, fruit- and vegetable
crops. Infestation of plants by thrips causes visible injuries on flowers, buds, fruits and young
leaves leading to a reduction in yield quality and quantity. These injuries are caused by both
thrips feeding, egg-laying and due to the reaction of plant tissue towards injected thrips
saliva. Thrips also serves as a viral vector in transmitting the tomato spotted wilt virus
(TSWV) and the maize chlorotic mottle virus (MCMV). As a result of legislation demanding
reduced pesticide usage, thrips resistance has become one of the most important breeding
targets in vegetable crops.
Research goals:
This project aims to facilitate the identification of genetic resistance to thrips. We are
planning to achieve this by:
1) Predicting and identifying proteins that are released into plants by thrips during infestation.
2) Isolating/cloning the corresponding genes and evaluate their effect on plant immunity in
plants that we genetically modify to express these thrips genes.
3) Encoded thrips proteins that show effect on plant immunity (i.e. effector proteins) are used
to fish out interacting plant proteins (i.e. effector targets).
4) We will then study how thrips effectors manipulate these interacting plant proteins to
perturb plant immunity and then try to envisage/develop plants with improved resistance to
thrips.
Available MSc internships (2015):
1) Development of thrips bioassay to screen for putative effectors: Techniques: gene
cloning, over expression of thrips genes in plants by agrobacterium infiltration, thrips
infestations and data analysis.
2) Silencing of thrips genes by feeding thrips on genetically modified plants:
Techniques: PCR, DNA cloning, gene expression by agrobacterium infiltration, virus
induced gene-silencing (VIGS), gene expression studies by Real-Time PCR.
3) Effect of thrips secretions on plant immunity: Techniques: collection of thrips
secretions and injection in plant leaves, diverse assays that monitor the activation of
plant defenses like Real-Time PCR, induction of chlorosis and defense hormones, callose
deposition and cell death.
Contact information:
Dr. ir. Ahmed Abd-El-Haliem, Departement of Plant Physiology, [email protected], Tel: 0205257891
Several research projects proposed by plant breeding companies: you choose!
Master programma:
Group/Research institute:
Contact and website:
Supervisors:
Green Life Sciences/ Life Sciences/ Biomedical Sciences
Green Student Lab; Swammerdam Institute for Life Sciences
[email protected]; www.greenstudentlab.nl
Rob Dekker [email protected]; Geneviève Girard [email protected]
The Green Student Lab is a new research training lab for and by students where the University of
Amsterdam is directly linked to companies in the green sector. It combines academic environment
and innovative questions from industry in a way that gives both students and companies many
advantages. The lab is primarily intended for students who do NOT aspire to an academic career, but
want to focus on an industrial career. The Green Student Lab officially opened in early 2014 and has harbored
already several successful student projects, trained many students and enthused all of them.
Students can choose a project from a portfolio, which they run in an
interdisciplinary team of 12-15 students (MSc, BSc and HBO). In the course of
their research, they gain experience in R&D from industry while developing
academic thinking. Finally, a combination of soft-skills courses, experienced
feedback on their personal and scientific skills, and interactions with GSL
supervisors and ad hoc experts leads them to gain a distinctive educational edge.
Expertise domains include plant sciences, RNA and molecular biology,
microbiology, genomics and genetics.
Projects so far have tackled issues such as:

Identifying viruses in plant samples by sequencing small RNAs derived from plant defense mechanisms;

Discovering and comparing micro RNAs between different tissues of a crop of interest;

Investigating the genetic background of an intriguing and commercially valuable novel crop phenotype;

Discovery of a harmful virus in commercially important crops.
More projects are available to interested students in partnership with companies such
as Rijk Zwaan, NakTuinbouw, Syngenta, Bejo Zaden B.V., Enza Zaden, etc.
Techniques





DNA + RNA Microarray technology
DNA + RNA + small-RNA 3rd generation sequencing (gDNA-seq + RNA-seq)
Basic + advanced Applied Bioinformatics
Mass spectrometry
Advanced microscopy
Study of the interaction between tomato proteins and Geminivirus replication
initiation protein
Masterprogramma: Green Life Sciences
Group/Research institute: Molecular Plant Pathology, SILS
Project supervisor: Marcel Prins, [email protected]
Daily supervisor: Francesca Maio, [email protected], 020-5258415
Whitefly-transmitted Geminiviruses are one of
the most important classes of plant viruses that
cause significant yield losses in economically
important crops like tomato (in the figure e.g. the
symptoms of the Tomato yellow curl leaf virus on
tomato leaves are shown), pepper, cotton and
soybean in different parts of the world.
Geminiviruses have small single-strand DNA genomes with limited coding capacities.
They encode for only 6-7 proteins: among them the Replication initiation protein
(Rep) is the only one essential for the viral DNA replication and the Replication
Enhancer protein (REn), as the name already suggests is not essential, yet plays an
important role in the efficiency of the DNA replication cycle. Geminivirus DNA
replication takes place in the nucleus of the host plant and it has been shown that
neither the Rep, nor the REn proteins display any enzymatic activities themselves, but
that the virus entirely relies on plant proteins with such activities that are part of the
normal plant DNA replication machinery. These protein complexes are recruited to
the viral DNA by the Rep protein.
Earlier studies in literature already indicated several proteins to associate with Rep or
REn, but we aim to identify additional components interacting with these two viral
proteins. One system we plan to use in our study is the expression of the viral protein
Rep linked to a tag (a short aminoacidic sequence or a fluorescent protein, such as
flag or GFP) in tomato protoplasts (plant cells without the cell wall) and the analysis
of the plant proteins pulled down together with Rep through mass spectrometry.
The internship project involves the performing of DNA plasmids isolation, protoplasts
isolation from tomato in vitro culture and transfection of the protoplasts with plasmids
containing Rep gene and tomato interacting genes. The interaction between the viral
and plant proteins will be analyzed by fluorescence microscopy and coimmunoprecipitation assay. The student will learn how to study and identify proteinprotein interaction in vivo and will be involved in the preparation of the mass
spectrometry experiment aimed to discover new plant components that associate with
the viral replication complex.
Techniques to be used:
tomato protoplasts isolation and transfection, protein affinity purification, DNA
plasmid isolation, cloning, SDS PAGE/western blot, BiFC
Recommended reading:
•
•
Hanley-Bowdoin L, Bejarano ER, Robertson D, Mansoor S (2013) Nat Rev Microbiol
11:777-788
Berggård T, Linse S, James P (2007) Proteomics 7: 2833–2842
Transcription factors regulating virulence in the pathogen Fusarium oxysporum
Some fungi are able to infect plants. To do this, the fungus needs specialized proteins,
for example to suppress plant defence responses and recruit nutrients. In the tomato
pathogen Fusarium oxysporum, the genes coding for these proteins are located on one
small chromosome. This chromosome can be transferred horizontally (i.e. not via a
sexual interaction) to other F. oxysporum strains. In this way, a previously nonpathogenic strain can turn into a pathogen.
Take this
pathogenicity
chromosome!
Yeah
…but how
do I switch
it on?
We know that the genes on the pathogenicity chromosome are strongly upregulated
during infection, especially ‘effector genes’; genes coding for small, virulence
proteins. Previously, we have identified a transcription factor on one of the ‘normal’
chromosomes, Sge1, that is involved in the expression of effector genes. Recently, we
found that one of the transcription factors on the mobile chromosome, Ftf1, is also
involved in effector expression.
Reasearch questions
1) How do the transcription factors Ftf1 and Sge1 work together?
2) Where do Ftf1 and Sge1 bind on the genome?
Strategy
1) If we overexpress FTF1 or SGE1, we can induce the expression of effector genes
even without the presence of the plant (the host). If we delete SGE1, the fungus
cannot express effector genes anymore. What if we knock-out SGE1, and
overexpress FTF1 at the same time? Is Ftf1 then able to by-pass Sge1 and induce
the expression of effectors anyway? Or do both need to be present? In a similar
approach, we will create and test overexpression of SGE1 in a FTF2 knock-out.
2) To find out where Ftf1 and Sge1 bind on the genome, we will express tagged
versions of the proteins in Fusarium and test their functionality. With these
Fusarium transformants we will do ChIP (Chromatin Immuno Precipitation) to see
if we can pull down the effector promoters.
Techniques
1) Fusarium transformation, DNA isolation from Fusarium, RNA isolation, cDNA
synthesis, qPCR, bioassays on tomato.
2) Cloning, Fusarium transformation, microscopy, western blot, bioassays on tomato,
ChIP, qPCR.
Let us know if you are interested or have questions and/or ideas:
[email protected] (supervisor) or [email protected] (examinor).
Location is key: PIN localization in salt stressed plants. Abiotic stress, Molecular biology, Microscopy The section of Plant Physiology, SILS, Testerink lab. Ruud Korver, MSc [email protected] or [email protected] for more information on the project. See also http://www.uva.nl/profile/c.s.testerink Techniques: Among others the student will use Confocal microscopy, Q/RT-­‐PCR and will generate new A. thaliana fluorescent lines. When a plant grows in a saline soil or the root encounters a local increase of salt in the soil different salt tolerance mechanisms kick in. One of these mechanisms is the recently discovered halotropism (Galvan-­‐Ampudia et al., 2013). Halotropism is the alteration of root growth direction under influence of increased salt concentrations in the soil. This alteration of root growth direction is caused by an asymmetrical auxin distribution in the root. This is in turn caused by the internalization of the auxin efflux carrier PIN2. In the root, auxin is transported by different PIN proteins. PIN3, PIN1 and PIN4 proteins transport auxin to the root tip through the stele. From there the PIN2 proteins transport the auxin upwards again through the epidermal and cortical cells. This directional transport is possible because of the uneven distribution of the PIN proteins on the plasma membrane (Wiśniewska et al., 2006). For example, in the epidermal cells the PIN2 proteins are located on the apical side of the plasma membrane in a normal unstressed cell and this facilitates transport back up into the root. During salt stress situations the auxin flow is altered, this indicates changes in the expression or localization of the PIN proteins. In order to fully understand halotropism it is important to know the exact localization of the different PIN proteins in the different cell types. In this project the student will study the localization and expression of different PIN proteins in the salt stressed Arabidopsis thaliana root using confocal microscopy and other techniques. Recommended reading: Galvan-­‐Ampudia, C. S., Julkowska, M. M., Darwish, E., Gandullo, J., Korver, R. A., Brunoud, G., ... & Testerink, C. (2013). Halotropism is a response of plant roots to avoid a saline environment. Current Biology, 23(20), 2044-­‐2050. Wiśniewska, J., Xu, J., Seifertová, D., Brewer, P. B., Růžička, K., Blilou, I., ... & Friml, J. (2006). Polar PIN localization directs auxin flow in plants. Science, 312(5775), 883-­‐883. Interests: Group: Daily Supervisor: Contact: Developing markers to identify a plant pathogen’s host
Fusarium oxysporum is a fungus that resides in the soil and is able to infect a large variety
of plants, causing wilt disease. The species as a whole is able to colonize the xylem vessels
of more than 100 different plant species, but each individual isolate is highly restricted to
one host. Naturally, with such a broad host range, much diversity exists also amongst
strains of F. oxysporum. The molecular arms races between Fusarium and different host
plants leads to selection for sequence variation in virulence or “effector” genes.
Additionally, we have shown that the chromosome that contains all the effector genes in a
tomato-infecting strain can be transferred between different F. oxysporum strains, making
the unravelling of this puzzle even more interesting.
In this project, we will try to develop genetic markers that can reliably predict whether an
unknown isolate infects either a cucumber, melon, watermelon, tomato or Brassicaceae
(e.g. Arabidopsis) plant, by:
-­‐
Screening for presence and expression of known effector homologs and (predicted)
virulence genes
-­‐
Collaborate with companies to test real unknown pathogenic strains from the field
or greenhouse and identify their host.
-­‐
Executing pathogenicity bio-assays
The goal of the research is to eventually pinpoint new effector proteins and hopefully
reconstruct part of the evolution of this remarkable plant pathogen.
Techniques involved: Plant infection assays (cucumber/watermelon/Arabidopsis), genetic
modification of fungi, DNA/RNA isolation, comparative genomics, (RT-)PCR, microbiological
lab techniques, possibly some bioinformatics.
Group:
Examiner:
Supervisor:
Molecular Plant Pathology
Martijn Rep
Peter van Dam
If you are interested in this project or have a question about it, please send me an email:
[email protected].
Filling in the gaps in SnRK2.4 signaling pathway
In order to adapt to unfavorable environments, plants need a wide range of signaling cascades transmitting
information about external factors to switch on protective mechanisms. One on the serious problems for plants is high
salinity. There are many factors responsible for salt signaling. In our group we focus on SnRK2.4 and SnRK2.10
protein kinases, which are activated by autophosphorylation within the first minute of applying salt stress. It has been
shown that they contribute to maintain Root System Architecture (RSA) in the presence of salt stress, but the molecular
mechanism behind it is so far unknown. Based on the mode of action of other kinases from the SnRK2 family, we
hypothesize that there is a protein phosphatase upstream of SnRK2.4 and 2.10 keeping them dephosphorylated and
inactive in control conditions. Application of salt disrupts this interaction, allowing SnRK2.4 and 2.10 to
autophosphorylate and phosphorylate their downstream targets. In order to identify up- and downstream targets of
SnRK2.4, we performed co-purification of TAP-tagged SnRK2.4 coupled with mass-spec analysis. We identified 7 very
promising downstream candidates.
Aims of this project are:
1.Confirm physical interaction of identified targets with SnRK2.4
2.Verify whether identified proteins are phosphorylated by SnRK2.4
3. Analyze the role of the confirmed SnRK2.4 targets in salt stress
Recommended reading:
1.
2.
McLoughlin, F., et al., The Snf1-related protein kinases SnRK2.4 and SnRK2.10 are involved in maintenance
of root system architecture during salt stress. Plant Journal, 2012. 72 (3): p. 436-449.
Van Leene, J., et al., An improved toolbox to unravel the plant cellular machinery by tandem affinity
purification of Arabidopsis protein complexes. Nat Protoc, 2015. 10(1): p. 169-87.
Technical skills/methods: molecular cloning, recombinant protein expression and purification, in-vitro kinase activity
assay, confocal microscopy, salt tolerance experiments
Availability: from November 2015 (min. 6 months)
Contact person: Dorota Kawa ([email protected]), Christa Testerink ([email protected])
More information about the research group: http://www.uva.nl/profile/c.s.testerink
https://www.facebook.com/ChristaTesterinksLab
Deciphering the tools of a plant pathogen:
How do Fusarium effectors suppress plant immunity?
Background:
Fusarium oxysporum is a devastating plant pathogen that can infect more than 100
different crop plant species. For successful infection, the pathogen needs effectors:
these are small proteins that are secreted inside the plant and help the pathogen to
colonize the plant, for example by suppressing plant immunity. So far 15-20 effectors
are identified in Fusarium oxysporum, but how they work to help infection remains a
complete mystery.
Questions:
1) Which Fusarium effectors are essential for infection?
2) Which effectors can suppress basal immunity?
Approach:
To answer question (1), Fusarium strains will be designed that are knocked-out for
single effectors. This will involve molecular biology techniques, such as PCR
amplification, molecular cloning into a plasmid, and transformation of the knock-out
construct into Fusarium. All these techniques are routinely used in the MPP
department.
The effector-KO strains will be then tested for virulence, by infecting plants and
observing the disease symptoms. Additionally, the colonization of Fusarium can be
followed by fluorescence microscopy (see picture).
To answer question (2), the effectors will be directly expressed inside plant cells (in
N. benthamiana, a relative of wild tobacco). Then plant immunity will be induced by
adding purified molecules (flagellin and chitin), and tested which effectors can
suppress immune responses. Two types of immune responses can be easily observed:
a rapid response (activation of protein kinases), and a late response (strengthening of
cell wall).
For this sub-project, 6 effectors have already been cloned in the right vectors, so that
the functional analysis can be directly started. Additional effectors could be included
if a student would like to.
From left-to-right: healthy and infected Arabidopsis plants, GFP-labeled Fusarium growing inside
roots.
Group:
Examiner:
Supervisor:
Molecular Plant Pathology
Martijn Rep ([email protected])
Nico Tintor ([email protected])
Please contact us if you have any questions!
Controlling bacterial invasion in Arabidopsis thaliana
Master program:
Availability:
Group/Research institute:
Contact person:
e-mail:
Green Life Sciences
Academic year 2015-2016, preferably 5+ months
Molecular Plant Pathology/Swammerdam Institute for Life
Sciences
Marieke van Hulten (Postdoc) or Harrold van den Burg (PI)
m.h.a.vanhulten[at]uva.nl; h.a.vandenburg[at]uva.nl
The bacteria Xanthomonas campestris pv. campestris (Xcc) is the causal agent of Back Rot, the most
important disease of cabbage (Brassica) crops worldwide. Xcc primarily enters the plant through
specialized organs on the leaf margins, called hydathodes, and spreads through the vasculature of the
plant. This colonization mechanism of Xcc is in contrasts to the closely related Xanthomonas
campestris pv. vesicatoria and Xanthomonas campestris pv. raphani, which both enter the plant leaf
via stomatal openings. Most studies on Xcc focus on vascular resistance mechanisms that take place
once the bacteria is inside the plant and information on earlier stages of plant colonization is severely
lacking. In our project we are especially interested in those earlier stages. We want to know if plants
are capable to control bacterial invasion and we want to elucidate the molecular mechanisms involved.
This will be done in close collaboration with two companies in the green sector. In order to identify
and characterize genes involved in resistance against Xcc we will make use of the wealth of
information available for the model plant Arabidopsis thaliana, a member of the Brassicaceae family.
Hereto we will screen a natural Arabidopsis population for different resistance mechanisms and follow
forward genetics approaches using mutagenized plants. Subsequently, we will map the genes involved,
followed by in-depth characterization of the candidate genes thus found.
Within this project, different aspects can be studied, depending of the stage of the project and
depending on your own interest and background. To study bacterial invasion in more detail, we aim to
modify our bacterial pathogen so that it expresses reporter genes, allowing us to follow the pathogen
over time while it colonizes different plant tissues. We are in the process of generating bacterial strains
that carry reporter genes such as GUS, LUX or GFP and setting up disease assays with them.
Furthermore, by individually deleting “known” virulence genes in the bacterial genome their
requirement for invading host plants can be tested.
Used techniques:
• Cloning and sequencing
• Bacterial and plant transformations
• Disease assays
• Reporter gene (GUS, GFP, LUX) expression
• Forward genetics (e.g. EMS-mutagenesis)
• Genome wide association
Is it physical separation sufficient to explain specialization?
Supervisor Dr. Aldana Ramirez, E-mail: [email protected]
Postdoctoral Researcher at Laboratory of Plant Physiology, Science Park tel. 020
525 7891. Alternative contact Robert Schuurink
Project description
Isoprenoid
compounds,
such
as
monoterpenes, diterpenes, carotenoids,
tocopherol, plastoquinone as well as the
prenyl moiety of chlorophyl, play important
and diverse roles in the primary and
secondary metabolism of plants. In spite of
their
tremendous
diversity,
these
compounds are all derived from two simple
basic building blocks with 5 carbon atoms
(IPP and DMAPP), produced in the plastids
via the MEP pathway. The first step in the
biosynthesis of plastidial IPP and DMAPP is
catalyzed by the enzyme 1-deoxy-D-xylulose 5-phosphate synthase (DXS), for which
three divergent isoforms are present in tomato, however only two seem to be
functional (DXS1 and DXS2). In terms of expression, DXS1 is more ubiquitously
expressed whereas DXS2 shows predominant expression in trichomes, which
explains their non-overlapping functions (primary vs. secondary metabolism).
Interestingly in addition to that, we have observed that the two proteins present a
different sub-organelle distribution (spotted vs. smooth distribution, see figure).
Giving these combined observations we hypothesize that DXS1 and DXS2
specialized functions might not only be explained by the differential tissue
distribution, but also being physically and/or spatially separated within the chloroplast
which in turn could lead to being subjected to differential regulations in terms of
protein-protein interactions. In consequence, the objectives of this project are: (i) to
determine whether there is interaction between DXS and other members of the
pathway, (ii) whether this interaction is different for the two isoforms, and (iii) to
determine whether the two isoforms interact with (similar or different) regulatory
elements. Knowing this will enable us to understand how trichomes achieve
allocation of precursors to so many different specialized products and to set ground
knowledge for the potential exploitation of these natural cell factories of important
botanical metabolites.
Techniques
1- Conventional cloning
2- Golden Gate based cloning
3- Protein complexes formation by Yeast two hybrid (Y2H)
Does change of location result in a different distribution?
Supervisor Dr. Aldana Ramirez, E-mail: [email protected]
Postdoctoral Researcher at Laboratory of Plant Physiology, Science Park tel. 020
525 7891. Alternative contact Robert Schuurink
Project description:
Glandular trichomes are specialized hairs found on the
surface of some vascular plants that play important roles in
the interactions between the plants and their environment.
Besides being responsible for the production of essential
oils and medically relevant products, trichomes also
produce substances like terpenoids that have evolved to
provide plants with protection against phytopathogenes.
In the cultivated tomato, terpenoids are produced in
significant amounts by type VI glandular trichomes via the
plastidial MEP (2-C-methyl-D-erythritol 4-phosphate)
pathway and the cytosolic MVA (mevalonate) pathway.
Although our preliminary research in N. benthamiana
shows the expected sub-cellular distribution of the different
members of the MEP and MVA pathways (plastids vs.
cytosol respectively), the question of whether this distribution will still be respected in
a different tissue (glandular trichomes) still remains to be demonstrated.
Particle bombardment is a powerful tool for transient expression experiments, given
that it allows for the efficient introduction of multiple genes by just coating the gold
particles with different constructs carrying different genes (i.e. gene of interest and a
marker gene linked to different fluorescent proteins). The outcome of this transient
expression can be evaluated two days after the introduction of the genes, by using
confocal microscopy. In this project we aim to combine these two potent techniques
to help us elucidate the exact sub-cellular and sub-organelle localization of the
different enzymes involved in the synthesis of precursor of important botanical
insecticides in the trichomes of tomato.
Objective:
To determination of sub-cellular ditribution of enzymes involved in the MEP and MVA
pathways in tomato glandular trichomes.
Techniques
1-Basic cloning and transformation techniques
2-Transient tomato trichome transformation by particle bombardment
3- Confocal Microscopy
'NLR-­‐type immune receptors: Investigating the link between DNA-­‐damage and plant immunity' Dr. Marijn Knip and Dr. Frank Takken Group/Research institute: Contact person: Supervisor: Molecular Plant Pathology, SILS Dr. Frank Takken, [email protected], T: 0205257795 Dr. Marijn Knip, [email protected] Like animals, plants can also get sick, and therefore possess a complex immune system. Both animals and plants can recognize pathogens with dedicated receptors. NLR-­‐proteins are a class of immune receptors that are present in both animals and plants. These receptors are the most important type of immune-­‐
receptors in plants, and play a key role in recognizing and orchestrating the immune response in reaction to pathogens. Surprisingly, little is known of how these proteins function. Activation of NLR proteins following pathogen recognition results in a conformational change. We recently reported (Fenyk et al. 2015) that this conformational change allows them to bind to plant DNA; a finding that agrees with the observation that these proteins are shuttling between cytoplasm and nucleus. It is not known, however, how these proteins elicit immune responses after being activated and what the role of DNA binding is in this process. Recent insights into NLR protein function indicate that induction of DNA damage (single stranded ‘nicks’) could be involved in triggering the immune activation. We offer a MSc.-­‐internship in which the relationship between DNA-­‐damage induced by Rx1 (a well studied NLR protein from potato) and the induction of an immune response will be investigated. Thereto, a system will be set-­‐up in Nicotiana benthamian, in which Rx1 activation can be precisely controlled. This will allow us to be able to study the nature and timing of Rx1-­‐induced DNA damage and subsequent immune-­‐response induction. Among others, the following techniques will be employed: • Molecular cloning • Transient gene-­‐expression and creation of transgenic plants, for studying timed and synchronized responses • Studying the induction of immune pathways, using expression of known immune genes (qPCR), or the production of reactive oxygen species (ROS) • DNA-­‐damage assessment using COMET and TUNEL-­‐assays If you are interested in the topic, and/or curious about our research, feel free to contact us to discuss our work and the opportunity to work with us in a MSc. Internship. References Fenyk et al. (2015) J. Biol. Chem. pii: jbc.M115.672121 Ruy Kortbeek email: [email protected] Tel: 020-­‐5257891 Room: SP C2.208 Synthesis of defence compounds in wild tomato species Introduc<on Commercial tomato (Solanum lycopersicum) is one of the biggest and economically most important crops on the planet. While growing in open field or semi-­‐open greenhouses, plants are con8nuously under aDack of insects and small herbivores. The plant is damaged not only by feeding but more importantly because insects carry harmful viruses that quickly spread and can cause crop losses up to a 100%. Commercial tomato is this vulnerable because breeding programmes have always focused on fruit size, taste and yield while natural defence mechanisms have not been selected for. Instead, chemical treatments were heavily relied on to keep crop plants produc8ve. Defence in a trichome: The ‘wild’ ancestral tomato species (e.g. S. habrochaites and S. pennellii) are s8ll capable of effec8vely defending themselves by synthesizing specific secondary metabolites in glandular trichomes. These are hair-­‐like structures on the plant’s surface with one or more glandular cells on top. The cells act as biochemical factories producing vola8le and non-­‐vola8le defence compounds that can act repellent or toxins to pest insects or that aDract its natural enemy. Examples of such compounds are acylsugars and terpenoids, and both groups appear to be highly diverse across species and accessions. Me
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ginger oil
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THF
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Keywords : •  wild tomatoes •  secondary metabolites •  insect resistance •  molecular biology (S)-curcumene 3
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Aim: As we start to learn more about the biochemical nature and biological relevance of these compounds, the regulatory mechanisms behind their synthesis remains largely unknown. We observed that there can be great differences in the abundance of specific compounds and we are interested in how the produc8on is triggered and how the underlying genes are consequen8ally regulated. To inves8gate this, the project will involve biochemical screening of wild tomato lines in different (8me) condi8ons and analyse the produc8on of defence compounds and the regula8on of the underlying genes. Techniques : •  wild tomato trichome characterisa8on •  Gas Chromatography -­‐ Mass Spectrometry •  RNA isola8on and cDNA synthesis •  Quan8ta8ve real-­‐8me PCR (qPCR) Department: Plant Physiology Group: Petra Bleeker; Natural VariaNon in Metabolic Defence Pathways in Tomato Master: Green Life Sciences Ruy Kortbeek email: [email protected] Tel: 020-­‐5257891 Room: SP C2.208 Iden1fying novel defence metabolites from wild tomato species contribu1ng to thrips resistance. Introduc1on Commercial tomato (Solanum lycopersicum) is one of the biggest and economically most important crops on the planet. While growing in the open field or semi-­‐open greenhouses, plants are con8nuously under aDack of insects and small herbivores. These damage plant 8ssue by feeding but more importantly they are the vector of harmful viruses. An example of this is the invasive Western Flower thrips (Frankliniella occidentalis) which spreads tomato spoDed wild virus (TSWV), thereby causing huge crop losses. Commercial tomato is vulnerable to pests like the Western Flower thrips because breeding programmes always focussed on fruit size, taste and yield while natural defence mechanisms were not selected for and were consequently lost. Defence in a trichome: The ‘wild’ ancestral tomato species (e.g. S. habrochaites and S. pennellii) are s8ll capable of effec8vely defending themselves by synthesizing specific secondary metabolites in glandular trichomes. These are hair-­‐like structures on the plant’s surface with one or more glandular cells on top. The cells act as biochemical factories producing vola8le and non-­‐vola8le defence compounds that can act repellent or toxins to pest insects or that aDract its natural enemy. Examples of such compounds are acylsugars and terpenoids, and both appear to be highly diverse across species. If we find which compounds contribute to insect defence, we might be able to ‘re-­‐arm’ our commercial tomato in the field. Keywords : •  wild tomatoes •  secondary metabolites •  insect resistance •  ecology and sta8s8cs Aim: We recently measured the terpenoids and acylsugars in a wide collec8on of wild tomato accessions cons8tu8ng of 10 different species. Now, we are interested to know which of these accessions are naturally resistant to the Western Flower thrips. By connec8ng the phenotype to the metabolome we want to find novel defence compounds contribu8ng to insect resistance. During this project, the student will develop a method and screen the resistance of our tomato collec8on to thrips infesta8on. This phenotype will then be linked to the composi8on and abundance of secondary metabolites in the tomato trichomes. Next, puta8ve THF
+for + defence compounds will be verified their biological role in controlled 1a
bioassays. PTAD
Me
H
N
ginger oil
O
Techniques : •  phenotypic screen •  controlled bioassays with metabolites •  isola8on of secondary metabolites •  Liquid/Gas-­‐Chromatography-­‐Mass Spectrometry 1a+1b
KOH/EtOH
reflux
H
N
N
O
N
N
N
O
O
N
N
O
Me
H
Me
H
Me
H
Pd/C /benzene
+
150°
Department: Plant Physiology Group: Petra Bleeker; Natural VariaQon in Metabolic Defence Pathways in Tomato zingiberene 2
Master: Green Life Sciences (S)-curcumene 3
7a
Socializing during salt stress: With whom to interact? Studying the lipid binding affinity of Clathrin assembly-­‐proteins. Interests: Abiotic stress, Molecular biology, Biochemisrty Group: The section of Plant Physiology, SILS, Testerink lab. Daily Supervisor: Ruud Korver, MSc Contact: [email protected] or [email protected] for more information on the project. See also http://www.uva.nl/profile/c.s.testerink Techniques: Among others the student will use Fusion protein purification, Liposome binding assays, Gateway cloning and Western blots. Salinization of the soil causes an increase in crop losses worldwide and a decrease of arable land. In order to continue producing enough food to keep up with the growing human population advances have to be made in the tolerance of plants to saline soils. One of these advances was the discovery of a new plant tropism, called halotropism (Galvan-­‐Ampudia et al., 2013), which describes the change of root direction when encountering an increase in salt in the soil. The change of growth direction is caused by internalization of the PIN2 auxin efflux carrier. This happens on the side of the root closest tot the salt gradient and results in an asymmetrical auxin distribution. PIN2 is internalized through Clathrin-­‐Mediated Endocytosis (CME). CME is dependent on Phosphatidic Acid (PA) produced by PLDζ. Recently, in a study investigating proteins that bind PA shortly after salt stress, two clathrin assembly proteins were found (McLoughlin et al., 2013). These proteins are potentially involved in recruiting clathrin to the plasma membrane. Therefore, it is important to know their phospholipid binding affinity. During this project the student will study the phospholipid binding affinity of (parts of) clathrin assembly proteins. This can be achieved by cloning different constructs with fusion proteins containing a GST tag and a (part of a) clathrin assembly protein. These proteins will then be transformed into E. coli and isolated from the bacteria. The isolated protein will be used for Liposome binding assays. In these assays liposomes with different compositions of phospholipids (PC, PE, PA, PI4P and PI(4,5)P) are created and the amount of protein that binds these liposomes is determined. Recommended reading: Galvan-­‐Ampudia, C. S., Julkowska, M. M., Darwish, E., Gandullo, J., Korver, R. A., Brunoud, G., ... & Testerink, C. (2013). Halotropism is a response of plant roots to avoid a saline environment. Current Biology, 23(20), 2044-­‐2050. McLoughlin, F., Arisz, S. A., Dekker, H. L., Kramer, G., de Koster, C. G., Haring, M. A., . . . & Testerink, C. (2013). Identification of novel candidate phosphatidic acid-­‐binding proteins involved in the salt-­‐stress response of Arabidopsis thaliana roots. Biochem J, 450(3), 573-­‐581. doi:10.1042/BJ20121639 What are the whiteflies molecular weapons in manipulating plant defenses? =
saliva effector protein
plant protein
Phloem =
Insects that are transmitting plant-­‐viruses from one plant to another are economical disasters for farmers. One such insect is Bemisia tabaci or whitefly. These flies transfer viruses through feeding. They penetrate the lower side of the leaf with their stylet (see figure), during this process saliva is excreted and the virus is transmitted. The whitefly is really clever; it produces proteins (effectors) within the saliva that bind to plant proteins. The binding between these proteins results in the plant being unable to detect the whitefly. Without this detection the whitefly can reproduce and become a plague very quickly. This project aims to identify the whitefly saliva effector proteins and the plant proteins it interacts with. This knowledge will provide the breeding industry with possibilities to breed plants that can detect the whitefly and reduces the virus transmission. So far a selection have been made of saliva proteins that could potentially be effector proteins. Questions; 1) Are the potentially effector proteins really effector proteins? Techniques used; various cloning techniques, confocal microscopy, whitefly bioassays 2A) Are there differences in expression levels of saliva genes in general and more specifically effector genes when whitefly is feeding on different plants? 2B) Which genes are differently expressed among whitefly colonies from around the world? Techniques used; RNA isolation, de novo assembly of RNA seq data and more 3) There is a difference in the feeding behavior and virus transmission between male and females. Is this difference due to their saliva proteins? Techniques used; dissection of whitefly, whitefly bioassays, mass-­‐spectrometry 4) If you want to learn more about protein:protein interactions, there is also a project possible were you will use the following techniques; various cloning techniques, confocal microscopy, yeast-­‐two-­‐hybrid assays, co-­‐IP Contact person: Paula van Kleeff, [email protected] Rob Schuurink, [email protected] Role of PIP2 in salt-­‐stress signalling and tolerance Supervisors: Dr. Teun Munnik and members of his lab (contact: [email protected]; tel: 020-­‐525 7763) Location: Section Plant Physiology, Swammerdam Institute for Life Sciences, UvA, Science Park 904. Techniques: Plant molecular biology (PCR; gene cloning; plasmid construction, isolation and transformation); confocal imaging – cell biology; GUS-­‐gene expression analysis; Arabidopsis Phenotyping -­‐ salt/drought tolerance; lipid-­‐ and protein biochemistry. Scientific background No toxic substance restricts plant growth more than salt does. Salt stress presents an increasing threat to agriculture. Among the various sources of soil salinity, irrigation combined with poor drainage is the most serious, because it represents losses of once productive agricultural land. The reason for this so-­‐called ‘secondary salinization’ (as opposed to primary salinization of seashore salty marshes) is simple: water will evaporate but salts remain, and accumulate in the soil. The stresses created by a high salt are two-­‐
fold. First, many of the salt ions are toxic to plant cells when present at high concentrations externally or internally. Typically, NaCl constitutes the majority of the salts. Sodium ions are toxic to most plants, and some plants are also inhibited by high concentrations of chloride ions. Second, high salt represents a water deficit or osmotic stress because of decreased osmotic potential in the soil solution. The mechanism of plant salt tolerance is a topic of intense research in plant biology.1 Lipid Signalling Salt stress triggers the formation of the signalling lipid, phosphatidylinositol bisphosphate (PIP2).2 In seedlings of Arabidopsis, this occurs within minutes. We know that the PIP2 is synthesised through phosphorylation of phosphatidylinositol monophosphate (PIP) by the enzyme, PIP kinase (PIPK). The model plant system, Arabidopsis thaliana encodes 11 PIPK genes.3 Using T-­‐DNA insertion knock-­‐out KO mutants, we have identified three PIPKs that are responsible for this salt stress-­‐triggered PIP2 synthesis. Recently, we obtained triple mutants, which are completely devoid of the salt-­‐stress activated PIP2 response. (Munnik lab, unpublished). These mutants now need to be characterized and phenotyped for their growth and salt-­‐ and drought tolerance. Using promotor-­‐GUS reporter lines, in planta gene expression of the PIPK genes will be studied, while PIP2-­‐biosensor lines will be used to monitor PIP2 in vivo using confocal imaging.2 Lastly, the student will be involved in cloning FP-­‐tagged fusions of the PIPKs for complementation and overexpression analyses. The objectives are to unravel the role of PIP2 in ion homeostasis and osmotic regulation, and to use this knowledge to engineer crop plants with enhanced salt tolerance. References: 1.
Zhu, J.-­‐K. 2007. Plant Salt Stress. eLS. http://dx.doi.org/10.1002/9780470015902.a0001300.pub2 2.
Van Leeuwen et al. (2007). Visualisation of PIP2 in the plasma membrane of suspension-­‐cultured tobacco BY-­‐2 cells and whole Arabidopsis seedlings. Plant J. 52, 1014-­‐1026. Munnik & Vermeer (2010) Osmotic stress-­‐induced phosphoinositide and inositolphosphate signalling in plants. Plant Cell Environ. 33, 655-­‐669. 3.
Improving drought-­‐ and heat-­‐stress tolerance in plants Supervisors: Dr. Teun Munnik and members of his lab (contact: [email protected]; tel: 020-­‐525 7763) Location: Section Plant Physiology, Swammerdam Institute for Life Sciences, UvA, Science Park 904. Techniques: Plant molecular biology (PCR; gene cloning; plasmid construction, isolation and transformation; promotor-­‐GUS expression); lipid biochemistry; protein biochemistry; confocal imaging/cell biology; Arabidopsis Phenotyping: development, drought-­‐ and/or heat-­‐stress tolerance. Scientific background: Drought and heat stress cause major yield losses in many different crop species every year. Breeding
programs for new tolerant varieties are diverse and tailored to specific needs of a particular crop. The
plant's response to drought- and heat stress, however, is complex, involving many physiological,
structural, morphological and biochemical changes, which interact with other environmental factors and
metabolic processes.
Recently, improved tolerance to drought (maize, canola, tobaco) or heat stress (Arabidopsis) has
been found by overexpression of a single PLC gene, which codes for phospholipase C, an enzyme that is
involved in phosphoinositide (PI) metabolism, regulating the level of various potential signalling
molecules that regulate signal transduction, gene expression, mRNA editing and chromatin remodelling,
but can also generate various precursors of important sugars related to stress tolerance.1-6 Whether any
PLC can cause tolerance, and whether any gene can increase both heat- and drought tolerance is unknown
and is the main topic of a research proposal that just have been granted by NWO. It is a collaboration
with the Bose Institute in India, who will focuss on rice, while Amsterdam will focus on the model plant,
Arabidopsis. We already have severtal mutants and overexpressors and need help to characterize them
further. The research is ment to explore the potential of PI metabolites and to discover novel ways to
improve and engineer stress tolerance in other crops.
Fig. 1: Model showing the main components of the PLC signalling pathway potentially generating drought-­‐ and heat tolerance. Abbreviations: PA, phosphatidic acid; PI, phosphatidylinositol; PIK, PI kinase; PIP, phosphatidylinositolphosphate; PIPK, PIP kinase; RFOs, raffinose family oligosaccharides;TIR1, auxin receptor; COI1, jamonate receptor. References: 1.
Munnik & Nielsen (2011) Green light for polyphosphoinositide signals in plants. Curr. Opin. Plant Biol. 14: 489-­‐497. 2.
Wang et al. (2008) Enhanced expression of phospholipase C 1 (ZmPLC1) improves drought tolerance in transgenic maize. Planta, 227, 1127-­‐1140. 3.
Georges et al. (2009) Over-­‐expression of Brassica napus phosphatidylinositol-­‐phospholipase C2 in canola induces significant changes in gene expression and phytohormone distribution patterns, enhances drought tolerance and promotes early flowering and maturation. Plant Cell Environ. 32: 1664-­‐16 81. 4.
Tripathy et al.. (2012) Characterization and functional validation of tobacco PLCδ for abiotic stress tolerance. Plant Molecular Biology Reporter 30: 488-­‐
497. 5.
Zheng et al. (2012) Phosphoinositide-­‐specific phospholipase C9 is involved in the thermotolerance of Arabidopsis. Plant J 69: 689-­‐700. 6.
Gao et al. (2014) Phosphoinositide-­‐specific phospholipase C isoform 3 (AtPLC3) and AtPLC9 function additionally to each other in thermotolerance in Arabidopsis thaliana. Plant Cell Physiol. 55:1873-­‐83 Negative effects of plant toxins on biological control organisms Masterprogramma: Green Life Sciences Group/Research institute: Molecular Biology; Institute for Biodiversity and Ecosystem Dynamics (IBED), UvA Contact person: Dr. Merijn Kant; [email protected]; Phone: (+31)-­‐20-­‐5257793 Supervisor: Dr. Livia Ataide; [email protected]; Phone: (+31)-­‐20-­‐5257739 Tomato is an important food commodity worldwide and it is relatively hostile host for many herbivores and microbes predominantly because of its glandular trichomes (leaf hairs). These are small chemical factories that produce and accumulate toxins and exude sticky substances. These toxins are considered important for the plant because they protect them against herbivory and in tomato they are roughly grouped as: (1) nitrogen-­‐containing metabolites, (2) phenolics and (3) terpenoids. Finally, some of these defensive substances are premade, i.e. present irrespective of the presence of a pest, while others are induced specifically by herbivory or infection. The spider mites Tetranychus urticae and T. evansi (see photos) are typical tomato pests worldwide and subpopulations have adapted to these tomato defenses in different manners. For example, the mite T. evansi evolved traits that enables it to suppress the biosynthesis of toxins by tomato, while other species evolved direct resistance to ingested tomato toxins. In general, herbivores resist plant toxins either via degradation, modification, excretion or sequestration and these adaptations are not mutually exclusive. There are some indications that spider mites can sequester toxins and it is a well-­‐
known phenomenon in many insect species. These sequestered toxins can be used by such herbivores as protection against their own natural enemies. We have preliminary evidence that this trait may interfere with the efficiency of biological control. Biological control is an environmentally friendly strategy to control pests not by using pesticides, but by using natural enemies of pests. These can be predators or parasitoids several of which you can commercially purchase to release in a greenhouse or open field as protection for a crop. Our data suggests that spider mites may store tomato toxins in their body in such a way that their biological control agents (predatory mites) do not like to eat them much anymore. In this research project we want to investigate: (i) if the performance of the predator is affected by defense compounds of tomato (Solanum lycopersicum) eaten by their prey and (ii) which compounds are responsible for this anti-­‐predator protection. Traditionally, the world of obtaining resistant plants via breeding and the world of biological control have largely operated separately. Only in recent years these two sectors began to exchange knowledge in order to optimize compatibility. The idea that plant-­‐resistance breeding can interfere directly with the suitability of a crop for biological control is something not yet taken into account when designing control strategies. Techniques: The project includes the use of Liquid Chromatography coupled to mass spectrometry (LC-­‐MS) and/or Gas Chromatography coupled to mass spectrometry (GC-­‐MS) and working with mites and plants (tomato, beans). References: Kant et al. (2015) Annals of Botany 115 (7): 1015-­‐1051 Alba et al. (2015) New Phytologist 205: 828-­‐840 Sarmento et al. (2011) Ecology Letters 14: 229-­‐236 Kant et al. (2008) Proceedings of the Royal Society B: Biological Sciences 275: 443-­‐452 Characterizing changes in chromatin landscape in Arabidopsis thaliana chromatin mutants Masterprogramma:
Group/Research institute:
Contact person:
Supervisor:
Green Life Sciences
Plant Development and (Epi)genetics/SILS
Dr. Maike Stam, [email protected], tel: 020-5257655
Mariliis Tark-Dame, [email protected]
This internship is a part of the STW project “Epigenetics meets targeted mutagenesis” that aims to improve the efficiency of targeted mutagenesis (ODM) by affecting chromatin structure. The results of the project will be used to generate improved crop varieties for plant breeding. For the STW project we have selected a number of candidate genes affecting chromatin structure. These genes encode for DNA methyltransferases, histone acetyl-­‐transferases, histone deacetylases, histone methyltransferases and other proteins known to be involved in establishing and maintaining the chromatin landscape. The effect of depletion of these proteins on chromatin structure is likely different. We hypothesize that a more accessible chromatin structure will enhance the efficiency of targeted mutagenesis. Results using Epi-­‐drugs suggest that our hypothesis might be true. This internship combines two interconnected sub-­‐projects: 1. Is the chromatin landscape altered in the candidate mutants? In order to verify that in the mutants of interest the chromatin structure is changed, a combination of biochemical and cytological tools will be used. Larger changes in chromatin structure and decondensation of heterochromatic regions will be visualized by staining DNA in the nucleus with DNA-­‐
binding dyes (DAPI). Western blotting or immunolabeling will be used to detect global changes in histone modifications. Changes in accessibility of euchromatic regions are often not visible under the microscope. Therefore a different approach needs to be used. Upon increased accessibility of genic regions, RNA polymerase can also initiate transcription in the gene body, leading to the formation of short, cryptic RNA molecules. Those RNA molecules can be detected on a gel using the Northern blotting technique. Setting up this method and selecting genes to be tested for cryptic transcripts is part of this internship. 2. Combining Arabidopsis thaliana chromatin mutants and ODM reporter lines. In the mutants ordered from the stock center the genes of interest are either inactivated by a T-­‐DNA insertion into the target gene or by the expression of an RNAi construct, a transgene encoding a double-­‐stranded RNA homologous to the target gene. It needs to be verified whether the lines ordered carry the transgenic insertion of interest and whether the expression of the gene of interest is downregulated. In order to answer these questions PCR and qPCR methods will be used. Mutants verified to be suitable are crossed with ODM reporter lines. In order to find Arabidopsis thaliana lines homozygous for chromatin mutations and the ODM reporter constructs, multiple rounds of genotyping and self-­‐crossing of plants will be necessary. Techniques DNA and RNA isolation, PCR, qPCR, fluorescent in situ hybridization, immunolabeling, fluorescent microscopy, Northern blotting, Western blotting, Flow cytometry, working with Arabidopsis thaliana Suggested reading and references Deal RB, Henikoff S. 2011. Histone variants and modifications in plant gene regulation. Curr Opin Plant Biol. 14:116-­‐22. Shu H, Wildhaber T, Siretskiy A, Gruissem W, Hennig L. 2012. Distinct modes of DNA accessibility in plant chromatin. Nat Commun. 3:1281. Li B, Gogol M, Carey M, Lee D, Seidel C, Workman JL. 2007. Combined action of PHD and chromo domains directs the Rpd3S HDAC to transcribed chromatin. Science. 316:1050-­‐4. Transcription factors regulating terpenoid biosynthesis in tomato trichomes Contact: Jiesen Xu, Plant Physiology, SILS, University of Amsterdam. Email: [email protected] Alternative contact: Robert Schuurink Introduction Tomato glandular trichomes are the major sites for volatile terpenoids production and emission. These monoterpenes and sesquiterpenes play a vital role in plant defense against herbivory. The biosynthetic pathway of terpenoids has been well studied in plants. However, the regulation of terpenoid biosysnthesis is still unclear, especially in secretory sites like glandular trichomes. There are few transcription factors (TFs) identified to be involved in regulation of terpenoid pathway in some species. In tomato, we found SlEOT2 and SlMYC1 can transactive promoters of terpene synthases based on transient transactivation assay in Nicotiana benthamiana. This suggests that SlEOT2 and SlMYC1 may be involved in terpenoid biosynthesis by regulating terpene synthases. To confirm this, we made transgenic lines with SlEOT2 or SlMYC1 silenced in tomato trichomes. Since SlMYC1 is also expressed in other tissues in tomato rather than trichomes, it may contribute to leaf volatile terpenoids production. Therefore, we also made transgenic lines with SlMYC1 silenced in all tissues. The aim of this project is to analyze these transgenic lines to investigate if the terpenoid biosysnthesis is affected. Hypothesis In tomato trichomes, transcripts level of terpene synthases and the amount of terpenoids decrease when SlEOT2 or SlMYC1 is silenced. Objectives 1, Quantitative Real-­‐time PCR (qRT-­‐PCR) to check the transcripts level of terpene synthases in SlEOT2 or SlMYC1 silencing lines 2, To check the internal volatile terpeneoids of the transgenic lines in tomato trichomes and leaf 3, To analyse the emission volatile terpenoids by trapping tomato plants Techniques DNA isolation, RNA isolation, qRT-­‐PCR, volatile terpenoids trapping and extraction, Gas Chromatography-­‐Mass Spectra (GC-­‐MS) analysis The very stupid caterpillar
At first glance, plants are easy food for hungry herbivores. While carnivores often have
to chase their pray and put up a struggle before they can eat, herbivores seem to have it
much easier because plant’s don’t run away. However, herbivores don’t get their meals
for free: plants do fight back. Plants can amongst others produce toxic compounds which
are ingested by the herbivore during feeding, but they also have much more subtle
defensive strategies: plants emit volatiles in response to herbivore damage thereby
attracting predators of the herbivores as part of an indirect defense. One main group of
plant volatiles is called green leaf volatiles (GLVs). These volatiles are known for their
characteristic smell of freshly cut grass. GLVs consist of C6-aldehydes, alcohols and
esters and are released in two different isomeric forms – (Z) and (E).
We recently showed that mechanically damaged leaves of the wild tobacco Nicotiana
attenuata release high amounts of (Z)-GLVs and low amounts of (E)-GLVs. However,
when the plant is attacked by its specialist herbivore, the tobacco hornworm (Manduca
sexta), the chemical composition of the GLV bouquet dramatically changes
characterized by a distinct shift from (Z)- to (E)-GLVs. This herbivore-induced change
in the (Z)/(E)-ratio attracts the generalist predator Geocoris spp., and the approaching
predator decreases the herbivore load on the plant by feeding on caterpillar eggs and
early instar larvae. In this specific tritrophic interaction (N. attenuata – M. sexta –
Geocoris), it is not the plant that is responsible for the conversion of the leaf aldehyde
(Z)-3-hexenal, but an enzyme present in the oral secretions of Manduca caterpillars(1).
These results are quite surprising since it shows that caterpillars are betraying
themselves by making the volatile bouquet more attractive to their own enemy. This
raises an important question: why does Manduca produce an enzyme that generates
volatiles which betray it to its own enemy i.e. why did evolution not select against it?
The occurrence of this hexenal-specific isomerase activity in at least two other
lepidopteran species suggests that it may have either vital or beneficial functions that
outweigh the caterpillar’s net costs of maintaining this enzyme. In a recent study we
could show that adult Manduca moths use the altered GLV-composition to determine
oviposition sites that are less likely to be attacked by predators or occupied by
competitors(2).
We have recently identified the enzyme that is responsible for the conversion from Z-3hexenal to E-2-hexenal by classical biochemical fractionation techniques and identified
it by LC-MS/MS.
References:
(1) S. Allmann & I.T. Baldwin (2010). Insects betray themselves in nature to predators
by rapid isomerization of green leaf volatiles. Science, 329 (5995), 1075-1078. doi:
10.1126/science.1191634S.
(2) S. Allmann, A. Späthe, S. Bisch-Knaden, M. Kallenbach, A. Reinecke, S. Sachse,
I.T. Baldwin & B.S. Hansson (2013). Feeding-induced rearrangement of green leaf
volatiles reduces moth oviposition. eLife, 2, e00421. doi: 10.7554/eLife.00421
Project 1: How can the caterpillar be so stupid to betray itself to its own enemy
For this project we will try to verify the following hypothesis:
The enzyme is involved in molting and is transcriptionally up- or downregulated
while caterpillars molt.
Molting is a manner in which insects routinely cast off
their exoskeleton to be able to grow.
We will grow Manduca sexta caterpillars on plants to
determine transcript levels of the (3Z:2E)-enal
isomerase throughout all larval life stages. We will
dissect caterpillars to determine expression values in
different tissues, isolate RNA, synthesize cDNA and
perform Q-RT-PCR analyses. We will grow caterpillars on artificial diet that
contains molting hormones to determine if this increases transcript levels of the
isomerase.
There is always room for your own ideas!
Project 2: Do other insects possess similar enzymes?
As (3Z):(2E)-enal isomerase activity has also been found in the oral secretions of
Spodoptera exigua and littoralis the protein sequence of the Manduca derived
isomerase will be used to search through insect genome/transcript databases for
homologs and we will carry out sequence analysis in several lepidopteran and
other crop eating species to investigate the across species diversification of the
enzyme.
We will furthermore collect spit
from several lepidopteran species
and test it for isomerase activity by
SPME-GC-ToF-MS. If homologous
isomerases can be identified we will
express (a subset of) these recombinant proteins
and assay them for isomerase activity towards (Z)-3-hexenal and other substrates
to explore functional diversification in addition to sequence diversification.
There is always room for your own ideas!
----------------------------------------------------------------------------------------------Contact details:
Silke Allmann
Plant Physiology
Science Park 904, room C2.208a
Email: [email protected]
Tel.: 020-5256236
Gene regulation by epigenetic mechanisms Masterprogramma: Group/Research institute: Contact person: Daily supervisor: Green Life Sciences Nuclear organisation group/Plant Dev & (Epi)genetics; Swammerdam Institute for Life Sciences Dr. Maike Stam, [email protected], tel: 020-­‐5257655 Rechien Bader, [email protected]. The Stam subgroup among others studies the role of epigenetics and chromatin structure in gene regulation. Epigenetic gene regulation refers to mitotically or meiotically heritable changes in gene expression that are mediated by changes in DNA methylation and chromatin structure. Epigenetic regulation is essential for the normal growth and development of an organism and the response to environmental cues. One of the systems we use for our studies are the maize B’ and B-­‐I alleles. B’ and B-­‐I have the same DNA sequence, but differ in expression level, DNA methylation pattern and chromatin structure. B’ is low expressed (light pigmented plants), while B-­‐I is high expressed (dark pigmented plants; see figure). The expression of the b1 gene is controlled by regulatory sequences 100 kb upstream of the gene. The B’ regulatory sequences are DNA hypermethylated, carry repressive histone marks and are silenced, while the B-­‐I regulatory sequences are hypomethylated, carry active histone modifications and enhance the transcription of the b1 gene in a tissue-­‐specific manner. Intriguingly, when combined by crossing, the B’ epiallele communicates in trans with the B-­‐I epiallele, changing B-­‐I into B’ in a mitotically and meiotically heritable manner. This trans-­‐inactivation process, which challenges the mendelian rules, is called paramutation. The regulatory sequences 100 kb upstream of the b1 coding region are required for this in trans inactivation of B-­‐I. In B’/B-­‐I F1 plants, the B-­‐I regulatory sequences become DNA methylated during plant development. This methylation process starts in the embryo and is only completed late in plant development. The molecular mechanisms underlying the in trans inactivation are indicated to require the small RNA-­‐directed DNA methylation (RdDM) pathway. Interestingly, our recent DNA methylation analyses at basepair resolution indicate that the sequences involved in the trans inactivation process carry DNA methylation patterns that deviate from those produced by the canonical RdDM pathway. This internship will focus on getting more insight into the mechanisms underlying the in trans silencing process by studying the deviating DNA methylation patterns at the sequences required for paramutation in more detail. Bisulfite sequencing will be performed on DNA isolated from plants carrying different b1 (epi)alleles and/or b1 transgenes, at different stages during development. Techniques The project amongst others involves recombinant DNA technology, DNA methylation analysis (bisulfite sequencing), (q)PCR and working with the crop plant maize. References o Chandler V.L., Stam M. (2004) Nat Rev Genet. 5, 532-­‐44. o Stam M., Mittelsten Scheid O. (2005) TIPS 10, 283-­‐290. o Stam M. (2009) Molecular Plant 2009 2(4): 578-­‐588. doi:10.1093/mp/ssp020. o Louwers M et al. (2009) The Plant Cell, 21, 832-­‐842. doi: 10.1105/tpc.108.064329 o Haring M et al. (2010) The Plant Journal, 63, 366-­‐378. o Belele et al. (2013) PLoS Genetics, 9(10), e1003773. doi: 10.1371/journal.pgen.1003773. o Gent et al (2014) The Plant Cell 26, 4903-­‐4917. doi/10.1105/tpc.114.130427. o Hovel I, Pearson NA, Stam M (2015) Semin Cell Dev Biol. In Press. doi: 10.1016/j.semcdb.2015.08.012. Validation of candidate regulatory sequences in Zea mays
Master program:
Green Life Sciences
Group/Research institute: Plant Development and (Epi)genetics /Swammerdam Institute for Life
Sciences (www.science.uva.nl/sils/nog)
Contact person:
Dr. Maike Stam, [email protected], tel: 020-5257655
Supervisor:
Blaise Weber, [email protected]
The Stam subgroup studies the role of epigenetic mechanisms in gene regulation. Epigenetic
gene regulation refers to changes in DNA methylation, histone modifications and other chromatin
marks that lead to variation in gene expression that are mitotically and/or meiotically heritable.
Those changes mediate their effect by altering the accessibility to relevant cis-regulatory elements
that are necessary to establish the correct gene expression patterns. An important class of cisregulatory elements are transcriptional enhancers, which can be located upstream or downstream
of their target gene, sometimes large distances (Mb) away. Whereas regulatory sequences are still
poorly characterized in plants, they have been extensively characterized in mammals. Active
enhancers are for example found to be associated with specific features such as co-localization
with specific histones marks, chromatin accessibility and the ability to physically contact their target
via the formation of chromatin loops. All together, these characteristics can be used at our
advantage to uncover new regulatory sequences in plants.
In the European EpiTRAITS consortium (www.epitraits.eu), we aim for genome-wide
identification of cis-regulatory sequences (enhancers) in the crop plant Zea mays. This ambitious
project relies on the integrative analysis of different epigenetic and chromatin marks (histone
acetylation, chromatin compaction, DNA methylation) as well as sequence conservation and RNA
expression levels. By identifying regions of overlap between these features, we have identified
numerous putative distant enhancer regions. To test if these regions indeed act as enhancers in
vivo, different approaches are being pursued, among others the project described below.
The internship project involves testing the
enhancer activity of candidate enhancers in
transient assays. Candidate enhancer sequences
will be cloned into a GUS reporter system
containing a minimal 35S promoter, a promoter
that needs enhancer sequences to promote
transcription. The constructs containing putative
enhancer elements will be bombarded into maize
tissue, which after incubation, will be checked for
reporter gene expression levels compared to that
mediated by control constructs. Sequences
promoting reporter gene expression will be
analyzed in more detail using other methods.
Techniques
The project involves recombinant DNA technology,
PCR analysis, Sanger sequencing, Particle
bombardment and reporter assays.
Figure 1: summary of the enhancer validation procedure.
Candidate enhancer are isolated from genomic DNA and inserted into a reporter
system. Plasmid DNA is loaded onto tungsten particles and bombarded at high
speed into maize tissue, followed by testing the reporter expression level.
References
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Andersson, R. et al. (2014). An atlas of active enhancers across human cell types and tissues. Nature 507, 455–461.
Louwers M, …and Stam M (2009) Tissue- and expression level-specific chromatin looping at maize b1 epialleles. The Plant
Cell 21(3), 21, 832-842. doi: 10.1105/tpc.108.064329
Mark L. Tucker, N.L. Using the VersaFluorTM Fluorometer to Quantitate GUS Expression in Plant Tissues Bombarded with
Biolistic® PDS-1000/He Particle Gun. Tech Note BioRad 2431.
Salvi, S. et al. (2007). Conserved noncoding genomic sequences associated with a flowering-time quantitative trait locus in
maize. Proc. Natl. Acad. Sci. 104, 11376–11381.
Shlyueva, D., Stampfel, G., and Stark, A. (2014). Transcriptional enhancers: from properties to genome-wide predictions. Nat.
Rev. Genet. 15, 272–286.
Stam, M., Belele, C., Dorweiler, J.E., and Chandler, V.L. (2002). Differential chromatin structure within a tandem array 100 kb
upstream of the maize b1 locus is associated with paramutation. Genes Dev. 16, 1906–1918.
Studer, A., Zhao, Q., Ross-Ibarra, J., and Doebley, J. (2011). Identification of a functional transposon insertion in the maize
domestication gene tb1. Nat. Genet. 43, 1160–1163.
Computational enhancer prediction from lncRNAs in maize
Master program:
Green Life Sciences, Bioinformatics & systems biology
Group/Research institute: Nuclear organisation/Plant Dev & (Epi)genetics; Swammerdam
Institute for Life Sciences
Contact person:
Dr. Maike Stam, [email protected], tel: 020-5257655
Supervisors:
Rurika Oka, [email protected]; Dr. Huub Hoefsloot,
[email protected]
One of the aims of the group of Maike Stam is to
identify transcriptional enhancers in the genome of the
crop plant maize. This is a subproject of the EU
EpiTRAITS consortium (www.epitraits.eu), which
aims to train young researchers in epigenetic gene
regulation and flowering in different model plants.
The internship project is focused on the genome-wide
computational prediction of enhancer sequences and
their characterisation in plants. The predictions will
enable and facilitate the downstream experimental
functional studies that are performed in the group.
Biological processes are tightly regulated and one of the major levels of regulation is at the gene
transcription level. The fine-tuning of gene expression during e.g. development, differentiation and
adaptation to environmental changes is accomplished in multiple ways, including the regulation by
transcriptional enhancers. Enhancers are located either up- or downstream of their target gene; in
mammals, they can be located up to ~1 Mb away of their target gene. This characteristic makes it
difficult to predict enhancer positions. For mammals, it has been shown that different features can
be used to identify and distinguish enhancers from the rest of the genome; for example low DNA
methylation, histone acetylation and chromosomal interactions between enhancers and their target
genes. It has also been shown that enhancers are transcribed and produce long non-coding RNAs
(lncRNAs) when they are active. It is assumed that plant enhancers have similar characteristics in
spite most of the knowledge on enhancers is derived from mammalian systems,.
In our study we use newly generated genome-wide next-generation sequencing (NGS) data from
two different maize tissues. Comparison of the data sets of these two tissues (see figure) allows us
to identify putative tissue-specific enhancers, which will then be experimentally tested for their
activities.
The internship involves the analysis of RNA-seq data and will focus on identifying lncRNAs in our
RNA-seq data sets. To capture putative tissue-specific enhancers, the student will also perform
differential expression analysis on the lncRNAs identified. Once lncRNAs and their genome
coordinates have been identified, their expression levels will be correlated to the expression levels
of nearby genes to predict their target genes.
Skills
The project will involve RNA-seq data analysis (Cufflinks, Trinity) and other bioinformatics tools
available on Linux. The student may use R, Python and/or Perl. Prior experience in Linux and
command line is an advantage.
References
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Zentner and Scacheri, J Biol Chem. 2012 287 30888-30896; DOI: 10.1074/jbc.R111.296491
Wang et al., GPB 2013 11 (3) 142-50; DOI:10.1016/j.gpb.2013.04.002
Shlyueva et al, Nat Rev Genet. 2014 15(4) 272-286; DOI: 10.1038/nrg3682
Wilken et al, Epigenetics Chromatin, 2015 8:8; DOI: 10.1186/1756-8935-8-8
Regulation of flowering through chromatin on the plant level
Till Bey and Paul Fransz, Plant (epi)genetics and development. Contact: email [email protected], phone 0205255153.
The floral transition
Plants precisely time the moment at which they start to flower to maximize reproductive success. This switch from
vegetative to reproductive growth is called the floral transition. But how is this important decision regulated?
Understanding the floral transition is not only relevant for agricultural applications but also a model to investigate
fundamental questions of biology. We are particularly interested in the floral transition from the genetic and
epigenetic perspective. It is known that a complex genetic network regulates this transition to integrate diverse
environmental and endogenous factors.
In the model plant Arabidopsis thaliana and presumably in other species as well, one of central players in this
network is the gene FLOWERING LOCUS T (FT). FT codes for a mobile protein that is expressed in the leaves and acts
at the apical meristem of the shoot. Expression of FT is sufficient to initiate flowering and loss-of-function mutants
flower with a severe delay.
How are signals integrated through FT? How does FT influence flowering time and
inflorescence architecture?
A multitude of transcription factors and chromatin proteins have been described to regulate the expression of FT.
We are interested in how the interplay between them can lead to a stable output in the form of FT expression level
and how FT expression levels of the entire organism are then integrated. We hypothesize that the chromatin state at
the FT locus plays a crucial role for robust regulation while showing large cell-to-cell variation.
Project plan
FT is silenced in most cells and only expressed in phloem cells, mainly at the tip of leafs. This is why we want to
investigate chromatin state and expression levels in single cells and preserved tissue to later identify cell type and
location of the cell in the leaf. We have established staining methods that allow us to visualize single genomic loci
simultaneously with histone modifications and chromatin associated proteins. Using confocal microscopy we then
record high resolution 3D images of nuclei and analyze colocalization of several signals. We aim to bring this
information together with results of growth experiments to understand FT regulation and action on the organismic
level.
The internship will be adapted to the current research carried out in our group. An internship on this project will
involve, depending on the wish and input of the student: recombinant DNA methods (molecular cloning, sitedirected mutagenesis), methods of plant genetics (transformation of plants, crossing, genotyping, phenotyping),
microscopy with advanced staining methods (detection of specific DNA sequences with FISH, immunodetection of
chromatin proteins, RNA detection using the MS2 system), and possibly some mathematical modelling.
[SOC1]
[FT]
Loci of FT and a FT regulator
in a nucleus stained by FISH.
Appearance of inflorescences after growth
in non-optimal conditions.
[AP1]
[LFY]
[CO]
[light]
Mathematical model of genes involved in flowering
time and floral meristem identity.