Download 2014-2015 Internship descriptions

 2014-­2015 Internship descriptions Track coordinator: Robert Schuurink Project proposal: Regulation of FT in chromatin
context
Till Bey and Paul Fransz, Nuclear Organization Group. Contact: [email protected]
Background: 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 it
is also a model to investigate fundamental questions of biology. We in particular are
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 many 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 meristem of the shoot.
Expression of FT is sufficient to initiate flowering and a loss-of-function mutants flower
with a severe delay.
Research question: How are multiple signals integrated at the FT
locus?
A multitude of transcription factors and chromatin proteins have been described to
activate or repress the expression of FT. We are interested in how the interplay between
them can lead to a coherent output in the form of FT expression level. We hypothesize
that the chromatin state at the FT locus plays a crucial role in the regulation. Influenced
by activators and repressors, we think it mediates their action and inhibits or favors
transcription.
Project plan
Only cells of specific cell type, mostly in the tip of the leaf, express FT. Thus in most cells
FT is silenced and is presumably in a different chromatin state. This makes it necessary to
investigate chromatin state and expression levels per each single cell while preserving the
tissue to correlate this with the position of cells in the leaf and their cell type.
We have established staining methods that allow us to visualize single genetic 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.
The internship will be adapted to fit with the current research carried out in our group.
Right now we are interested in assembling reporter gene constructs and tranforming
them into plants. This will allow us to monitor transcription in every single cell using the
RNA binding protein of the MS2 system. DNA constructs will be assembled using a
custom modular cloning system that has been developed in our group and that enables
flexible cloning of large endogenous plant sequences.
Combining RNA detection with observation of the FT locus allows us to directly assess the
influence of chromatin on expression for this gene. Moreover, we would like to use these
methods to demonstrate regulation of FT by additional transcription factors.
Methods
An internship on this project will involve recombinant DNA methods (molecular cloning,
site-directed mutagenesis), methods of plant genetics (transformation of plants, crossing,
genotyping, phenotyping), and microscopy with advanced staining methods (detection of
specific DNA sequences with FISH, immunodetection of chromatin proteins, RNA detection
using the MS2 system).
What is the role of plant defense hormone pathways in Arabidopsis – Fusarium interactions? Supervisor: Nico Tintor, [email protected]; alternative contact Martijn Rep Background: Fusarium oxysporum is a fungus that infects many different plant species, including the model plant Arabidopsis. Fusarium colonizes the plant roots, enters the vasculature and grows to the green plant tissues, thereby causing heavy disease symptoms. Currently it is little understood how plants defend themselves from this pathogen. In particular it is not clear where in the plant (root? vasculature? shoot?) immune responses are activated and which signaling pathways mediate the immune response. Aim of the project: The following plant defense signaling pathways will be analyzed for their role in Arabidopsis – Fusarium interaction: Jasmonate (JA), Ethylene (ET), Salicylic Acid (SA) are small molecules (hormones) that activate well-­‐
characterized signaling pathways. Phytoalexin deficient 4 (PAD4) is a nucleo-­‐cytoplasmic protein that is a key regulator of plant immunity. We wish to know, how (positively/negatively?) these regulators influence Arabidopsis interaction with adapted or non-­‐adapted Fusarium. Approach: 1) Which pathways activate/suppress plant defense?  plant infection assays We have now Arabidopsis lines that simultaneously knock-­‐out 2, 3 or all 4 of the above pathways. The student could test in plant infection assays, how these knock-­‐out plants respond to different (adapted and non-­‐adapted) Fusarium strains. This will identify which pathways activate or suppress defense to Fusarium, and also how they work together (crosstalk). 2) Which stages of the plant infection are influenced by JA/ET/SA/PAD4pathways?  microscopy Arabidopsis root infection can be easily monitored by fluorescence-­‐microscopy. For this we have fungal strains that are labeled with green-­‐fluorescent protein. This will reveal, which steps of plant infection (root colonization, entry into vasculature…) are affected in defense hormone KOs. 3) Where are JA/ET/SA/PAD4 pathways activated?  gene expression analysis This would include analysis of defense gene expression in different plant tissues by qRT-­‐PCR. Main techniques: Plant infection assays; in vivo imaging by fluorescent microscopy; gene expression analysis by quantitative RT-­‐PCR Green Life Science Internship Project
title: Identification of tomato genes that control homeologous recombination
The genetic variation in germplasm of food crops is limited and will soon become insufficient
to meet the demands of an increasing world population. Related wild species comprise crucial
sources of genetic variation, including resistance to pathogens or tolerance to changed
climate conditions. The transfer of genes from wild species to cultivated crops through
conventional crossing and backcrossing however is difficult and requires time-consuming
introgressive hybridization breeding. One of the major obstacles is the low transmission rate
of the desired combination of traits due to problems in homeologous recombination.
Extensive sequence divergence between species may hamper chromosome pairing and DNA
mismatch repair, two important processes during early meiosis. As a consequence the
number and position of recombination events between homeologous chromosomes is limited
compared to homologous recombination. A better control of homeologous recombination is
therefore crucial for plant breeders to improve the transfer of genes from wild to cultivated
species. Our aim is to identify (novel) genes that control homeologous recombination in
tomato (S. lycopersicum). Tomato is an important model crop to study meiosis, with excellent
cytogenetics and extensive genetic linkage maps. Initially we will focus on genes known to
affect recombination frequency in Arabidopsis, mouse and yeast: mlh1, mlh3, mus81, fancm,
hei10. The tomato orthologs of these genes have already been identified. At a later stage
during the project more genes of interest will be examined and tested.
Within this project we can supervise an interested master student who would like to do his/her
Green Life Sciences Internship. The goal of the GLS student project is to generate and
analyze transgenic plants for aberrant meiosis. The student will make overexpression and/or
knock-down (RNAi) constructs for the genes of interest, transform tomato plants and analyze
the meiotic phenotype of the transformants.
Techniques and tools:
- DNA cloning (generation of transgenic construct): various molecular biological
techniques, e.g. restriction digest, ligation, PCR
- plant transformation
- analysis of expression of the transgenic constructs: protein gel, western blot
- analysis of meiotic tissue of transformants: various immunocytological techniques,
e.g. spreading of meiocytes, immunofluorescent labeling, fluorescence microscopy
Supervisors:
- Esther de Boer ([email protected])
- Paul Fransz ([email protected])
Tomato Trichomes strategies to manipulate resourses.
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
Geranylgeranyl diphosphate synthases (GGPSs) are enzymes not only involved in
the biosynthesis of primary isoprenoid compounds (gibberellins, carotenoids,
chlorophyll), but also of volatiles produced by many plants in response to herbivory
(e.g.TMTT). Two GGPS genes with 55% sequence similarity have been reported in
tomato. LeGGPS1 is involved in the production of substrates for the biosynthesis of
volatile compounds (TMTT) in leaves, whereas LeGGPS2 is thought to be more
dedicated to the production of carotenoids in fruits and flowers. Keyword
interrogation of a tomato trichome EST database resulted in two new putative
GGPSs genes (named GGPS24 and GGPS86) present as full-length and in high
abundance (200-400 reads) compared to an, unexpectedly, incomplete and poorly
represented LeGGPS1 (2-40 reads) and the complete absence of LeGGPS2.
Phylogenetic analysis shows that GGPS24 shares a higher sequence similarity
(63%) with an Arabidopsis GGPS gene - suspected to have rather geranyl
diphosphate (GPS) than GGPS activity - than to the tomato GGPPs (38%). GGPS86,
on the other hand, shares between 56 and 62% sequence similarity not only with its
tomato counterparts but also with other plant GGPSs (pepper and sunflower).
Because the biosynthesis of GGPP is a branch point in the biosynthesis of mayor
isoprenoid compounds with diverse roles, the presence of different GGPS isoforms
with differential tissue expression, different subcellular localization, and/or differential
protein regulation would provide tissues in general and trichomes in particular with a
certain metabolic plasticity necessary to allocate resources where is needed.
Through this study we aim to unveil the complete tomato GGPP gene family and
shade some light on trichome metabolic fluxes towards distinct isoprenoids.
Objectives:
1-Unveil the tomato GGPS family.
2-Characterize all GGPPs found in terms of differential tissue expression.
3-Determine the cellular, sub-cellular (and maybe sub-compartment) localization of
all GGPSs within trichomes.
Techniques:
1-Basic cloning techniques.
2-Agrobacterium mediated transient transformation (ATTA).
3-Protoplast transfection
4-RTqPCR
6-Microscopy
Evolutionary Development
The genetic control of inflorescence architecture
Summary research topic:
Flowering plants exhibit an enormous variety in the way flowers are arranged on their inflorescence branches.
We have identified a gene from Petunia, EVERGREEN (EVG) that seems to have played a key role in the
evolution of cymose inflorescences (Rebocho et al. 2008). EVG stimulates outgrowth of inflorescence
meristems and indirectly regulates the floral meristem identity gene DOUBLE TOP (Souer et al. 1998/2008).
EVG presumably arose by duplication of a gene involved in embryonic meristem formation. How EVG
evolved from its ancestral gene is one of the questions we would like to address in this project. To
reconstruct the evolutionary events we will analyse the promoter and coding sequences of EVG and
homologous genes from other species as well as their expression patterns in diverse species. SISTER OF
EVG (SOE), the second gene that resulted from the presumed duplication, seems to have retained the
ancestral embyonic meristem function. We will compare the activity and specificity of EVG and SOE
inducible overexpression lines to determine if these two proteins diverged functionally.
Experimental techniques:
DNA/RNA isolation, cloning, sequencing, plant transformation, phylogenetic analysis, qPCR,
confocal/fluorescence microscopy, induction experiments, bioinformatics.
Supervisor:
Address:
Ronald Koes/Afke Sietsma
Vrije Universiteit, Institute of Molecular Cell Biology, Department of Genetics, de Boelelaan
1085, 1081 HV Amsterdam.
Email:
[email protected]
Starting date: After consultation
Length:
min. 24 weeks
Literature:
Rebocho, A. B., Bliek, M., Kusters, E., Castel, R., Procissi, A., Roobeek, I., Souer, E. and Koes, R. (2008). Role of
EVERGREEN in the development of the cymose petunia inflorescence. Dev Cell 15, 437-47.
Souer, E., Rebocho, A. B., Bliek, M., Kusters, E., de Bruin, R. A. and Koes, R. (2008). Patterning of Inflorescences and
Flowers by the F-Box Protein DOUBLE TOP and the LEAFY Homolog ABERRANT LEAF AND FLOWER of
Petunia. Plant Cell.
Souer, E., van der Krol, A., Kloos, D., Spelt, C., Bliek, M., Mol, J. and Koes, R. (1998). Genetic control of branching
pattern and floral identity during Petunia inflorescence development. Development 125, 733-42.
Hormones in Development
Linking inflorescence architecture with auxin biosynthesis and signalling
Summary research topic:
The EVERGREEN (EVG) gene of Petunia plays a crucial role in development of the
inflorescence branch (Rebocho et al. 2008). EVG seems to stimulate the outgrowth of the
inflorescence meristem. EVG encodes a for a homeodomain transcription factor. We have linked
EVG with sensing or synthesis of the plant hormone auxin. To explore the role of auxin in
inflorescence architecture we would like to analyse the genes activated by EVG; i.e. the
regulation and expression pattern. Also we have evidence that EVG influences the auxin
transport (PIN) proteins, these proteins have been excessively studied in Arabidopsis and we
would like to use this model for analysis of the behaviour of the PIN proteins upon ectopic
EVG expression. Also, it seems that EVG regulates the expression of the floral meristem
identity gene DOT (Souer et al. 1998/2008). In Arabidospis it has been shown that expression of
its homolog LFY is regulated by an auxin response factor ARF5/MP (Li et al., 2013) and EVG is
known to interact with members of the ARF-family (Y2H). We would like to see whether
ARF5/MP can also regulate the expression of DOT in Petunia.
Experimental techniques:
DNA/RNA isolation, cloning, sequencing, plant transformation, phylogenetic analysis, qPCR,
Bi-molecular Fluorescence Complentation (BiFC), fluorescence and confocal microscopy, ,
bioinformatics.
Supervisor:
Address:
Ronald Koes/Roeska Blankevoort
Vrije Universiteit, Institute of Molecular Cell Biology, Department of Genetics,
de Boelelaan 1085, 1081 HV Amsterdam.
Email:
[email protected]
Starting date: After consultation
Length:
min. 24 weeks
Literature:
LEAFY Controls Auxin Response Pathways in Floral Primordium
Formation Wuxing Li, Yun Zhou, Xing Liu, Peng Yu, Jerry D. Cohen and Elliot M. Meyerowitz
(9 April 2013) Science Signaling 6 (270), ra23. [DOI: 10.1126/scisignal.2003937]
Rebocho, A. B., Bliek, M., Kusters, E., Castel, R., Procissi, A., Roobeek, I., Souer, E. and Koes,
R. (2008). Role of EVERGREEN in the development of the cymose petunia inflorescence. Dev
Cell 15, 437-47.
Souer, E., Rebocho, A. B., Bliek, M., Kusters, E., de Bruin, R. A. and Koes, R. (2008). Patterning
of Inflorescences and Flowers by the F-Box Protein DOUBLE TOP and the LEAFY Homolog
ABERRANT LEAF AND FLOWER of Petunia. Plant Cell.
Souer, E., van der Krol, A., Kloos, D., Spelt, C., Bliek, M., Mol, J. and Koes, R. (1998). Genetic
control of branching pattern and floral identity during Petunia inflorescence development.
Development 125, 733-42.
Characterization of natural variation in the regulation of root stem cell activity in Arabidopsis thaliana In our group we study the natural variation in Root System Architecture in Arabidopsis thaliana. Variation in root development in control conditions was associated with allelic polymorphism in SET DOMAIN GROUP 2 (SDG2) gene, encoding protein with H3K4-­‐methyltransferase activity. SDG2 and has been described as a factor in root stem cell niche maintenance. The sdg2-­3 mutant loses cell identity and stem cell functioning during growth, resulting in shorter main root and decreased number of lateral roots (Yao et al, 2013). The importance of SDG2 in root growth and development was reported, but not the allelic variation that we observed. Within this project we would like to characterize natural variation in this gene in Arabidopsis thaliana. Project objectives: 1.Analysing accessions with different Root System Architecture in terms of: • SDG2 sequence polymorphism • SDG2 expression level • Root meristem size 2. Transforming the most promising accessions with quiescent center marker 3. Cloning SDG2 alleles from different accession 4. Complementation of sdg2-­‐3 phenotype with different SDG2 alleles Recommended papers: Yao, X., H. Feng, Y. Yu, A. Dong and W.-­‐H. Shen (2013). "SDG2-­‐Mediated H3K4 Methylation Is Required for Proper Arabidopsis Root Growth and Development." PLoS ONE 8(2): e56537. Meijon, M., S. B. Satbhai, T. Tsuchimatsu and W. Busch (2014). "Genome-­‐wide association study using cellular traits identifies a new regulator of root development in Arabidopsis." Nat Genet 46(1): 77-­‐81. Avalibility: from November 2014 (min. 6 months) Contact: Dorota Kawa : [email protected] Christa Testerink: [email protected] https://www.facebook.com/ChristaTesterinksLab http://www.uva.nl/over-­‐de-­‐uva/organisatie/medewerkers/content/t/e/c.s.testerink/c.s.testerink.html Controlling pathogenic bacterial invasion in Arabidopsis thaliana
Master program:
Availability:
Group/Research institute:
Contact person:
e-mail:
Green Life Sciences
Academic year 2014-2015, 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
Plants have a natural capacity to defend themselves against pathogens. But what makes a plant resistant
against a certain pathogen, while susceptible to others of the same species? And can we use that
information to breed crops with a durable enhanced disease resistance? Progress has already been made
towards understanding various plant defense mechanisms. However, we are interested in resistance
mechanisms that operate at the very early stages of colonization, of which not much is known.
Therefore we will embark on journey this year to elucidate the molecular mechanisms that are
available to a plant to control pathogenic bacterial invasion. 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 a bacterial pathogen during the start of the colonization process we will make use of
the wealth of information available for one of the host of this pathogen, the model plant Arabidopsis
thaliana. Hereto we will screen both natural populations 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.
For both screens, disease assays need to be set-up and tested. To study bacterial invasion in
more detail, we aim to modify our bacterial pathogen so that it expresses reporter genes, allowing
easier identification. Within this project, different aspects can be studied, depending of the stage of the
project and depending on your own interest and background. This project has only just started from
mid 2014. The emphasis in the first half of this academic year will lay on the generation of bacterial
strains that carry reporter genes such as GUS or GFP and setting up disease assays with them. To
ensure our results to be applicable to crop plants, setting up infectious conditions that are as close to the
natural situation as possible is vital. In the second half of this academic year we will shift towards
screening large populations of plants, both wild type and mutants, in order to identify genes involved in
controlling bacterial invasion.
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 intracellular translocation of Fusarium Avr2 controlled by the effector Six5?
Master internship Green life Sciences / Laboratory of Molecular Plant Pathology
Contact person: Dr Frank Takken, [email protected], +31205257795
Daily supervisor: Cao Lingxue, [email protected], +31205257683
Detailed programme of the traineeship period.
Plants contain Resistance proteins that can recognize specific pathogen-derived
“effector” proteins and subsequently initiate immune responses. Typically one
effector is recognized by one R protein. We found a deviation from this rule; Avr2
and Six5 from Fusarium oxysporum f.sp. lycopersici (Fol) are both required to trigger
I-2-mediated resistance in tomato. Deletion of either one of these effectors
compromised virulence in susceptible plants and avirulence on resistant I-2-tomato
plants. Avr2 and Six5 interact in the yeast-two-hybrid system as well as in planta as
shown by bimolecular fluorescence complementation (BiFC). The BiFC signal was
found to co-localise with plasmodesmata suggesting a role of Avr2/Six5 at these
channels. Our research is now focused on determining how Avr2/Six5 activate I-2
mediated defence responses and the possible role of plasmodesmata in this process. In
this project, the accumulation of Six5 at the plasma membrane and plasmodesmata
will be examined in both 35S::dspSix5 stable transgenic Arabidopsis thaliana plants
and following Agrobacterium-mediated transient transformation of Nicotiana
benthamiana leaves. Transgenic Avr2, I-2-breaking-Avr2 variants, Six5 and
Avr2/Six5 carrying Arabidopsis plants have been generated and their immune
responses to Arabidopsis-infecting Fusarium strain Fo5176 and vascular pathogen
Verticillium strain JR2 will be assessed in bioassays. This project should reveal the
function of Avr2 and Six5 at the plasmodesmata and provide mechanistic insight in
how they contribute to fungal virulence.
Knowledge, skills and competences to be acquired by the trainee at the end of the
traineeship
Knowledge: Insight in molecular plant pathology and specifically in the interaction
between Fusarium and tomato/Arabidopsis. Specific knowledge of the molecular
mechanisms underlying resistance and susceptibility of plants to vascular diseases
caused by filamentous fungi.
Skills:
• Experience with plant protein extraction methods (i.e. isolation of total
protein, plasma membrane protein fractions, soluble proteins etc.).
• Western blotting to detect proteins
• Agro mediated transient expression in N. benthamiana
• Arabidopsis genetics and genotyping (&PCR)
• Arabidopsis bioassays with Verticillium and Fusarium
Competencies:
Be able to designing a bioassay and perform genotyping of Arabidopsis.
Communicate and discuss the results with the direct supervisor and colleagues in the
same laboratory. Present the finding orally and in a scientific report to experts
working in the same research area.
How a stress pathway controls plant innate immune signaling. Introduction SUMO (small ubiquitin-­‐like modifier) is a post-­‐translational modification implicated in many biological processes. SUMOylation is essential in Arabidopsis, as null mutants of SUMO conjugation enzymes SAE1/SAE2 and SCE1 are embryo lethal. It was shown that plant stress conditions like a heat shock cold and drought trigger a global SUMO conjugation response correlated with protein damage response (reviewed in Mazur & Van den Burg, 2012). Interestingly, the SUMO conjugation enzymes were found to be targets of many pathogen effectors, and the plant hormones levels are deregulated in SUMO conjugation enzymes mutants. Research question: 1.What is the effect of plant hormones treatment (salicylic acid, jasmonic acid) on global SUMOylation in Arabidopsis? 2. What are the dynamics of SUMOylation during plant immunity/ biotic stress in Arabidopsis? Techniques to be used: • DNA techniques (traditional cloning, gateway cloning, sequencing) • Plant protein extraction • SDS-­‐PAGE • Western Blotting • Arabidopsis growing Recommended reading: •
•
Mazur, M.J. and van den Burg, H.A. (2012). Global SUMO proteome responses guide gene regulation, mRNA biogenesis, and plant stress responses. Front. Plant Sci. 3:215. doi: 10.3389 van den Burg, H.A, Kini, R., Schuurink, R. andTakken, F.L.W. (2010) Arabidopsis SUMO paralogs have distinct functions in development and innate immunity Plant Cell 22:1998-­‐
2016 Contact person: Harrold van den Burg: [email protected] (http://www.uva.nl/over-­‐de-­‐uva/organisatie/medewerkers/content/b/u/h.a.vandenburg/h.a.van-­‐den-­‐
burg.html) Characterizing changes in chromatin landscape in Arabidopsis thaliana chromatin mutants Masterprogramma: Group/Research institute: Contact person: Supervisor: Green Life Sciences Nuclear organisation group/ Plant Dev & (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 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. The overall aim of the internship is to test the effect of the depletion of various chromatin proteins on the chromatin landscape. To this purpose two main questions will be addressed: 1. Are the selected Arabidopsis thaliana chromatin mutants suitable for our studies? 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. 2. 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) or by treatments with specific DNA probes or antibodies (fluorescent in situ hybridization, immunolabeling techniques) to detect specific DNA regions or histone modifications, respectively. 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 RNA blotting technique. Selecting genes to be tested for cryptic transcripts is part of this internship. Techniques DNA and RNA isolation, PCR, qPCR, fluorescent in situ hybridization, immunolabeling, fluorescent microscopy, Northern blotting, 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. Tomato Trichomes as cell factories of diverse botanical metabolites
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 committed step in the biosynthesis of plastidial IPP and
DMAPP is catalyzed by the enzyme 1-deoxy-D-xylulose 5phosphate synthase (DXS), for which three divergent isoforms
are present in tomato. DXS1 and DXS2 have been shown to
have non-overlapping functions, manly due to their differential
tissue expression. DXS1 is more ubiquitously expressed whereas DXS2 shows
predominant expression in trichomes. Interestingly, although at considerably lower
levels, we recently discovered that DXS3 also is expressed in trichomes.
Giving their overlapping trichome expression and their rather divergent sequence
similarity (57%), we hypothesize that the DXS2 and 3 enzymes might have
specialized functions to meet the demands for different isoprenoids pools, either by
being physically separated (different specialized cells within the trichomes),
temporally (along trichome development) or by being subjected to a differential
regulation (protein-protein interactions). 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.
Objectives:
1-Determination of cellular, sub-cellular (and maybe sub-compartment) localization of
DXS2 and DXS3
2-Identification of interacting partners for DXS2 and DXS3.
3-Functional characterization of recombinant DXS3.
Techniques
1-Basic cloning techniques and in vitro protein expression
2-Agrobacterium mediated transient transformation (ATTA).
3-Protoplast transfection
4-Particle bombardment
5-Yeast 2 Hybrid (Y2H)
6-Microscopy
Flowering Gene regulation by epigenetics and chromosomal interactions Master program: Green Life Sciences Group/Research institute: Nuclear organisation group/Plant Dev & (epi)genetics group; Swammerdam Institute for Life Sciences Contact person: Dr. Maike Stam, [email protected], tel: 020-­‐5257655 Supervisor: Blaise Weber, [email protected] The Stam subgroup studies the role of epigenetic mechanisms and chromosomal interactions in gene regulation. Epigenetic gene regulation refers to changes in DNA methylation and histone modifications that lead to changes in gene expression that are mitotically and/or meiotically heritable. Chromosomal interactions refer to protein-­‐mediated interactions between e.g. the transcription start site (TSS) of a gene and cis-­‐acting regulatory sequences (enhancers). These regulatory sequences can be nearby, in higher eukaryotes gene regulation, however, also involves specific physical interactions between chromatin regions that are up to hundreds of kilo base pairs apart from each other on the same chromosome or even on different chromosomes. In the European EpiTRAITS consortium (www.epitraits.eu), we aim to identify chromosomal interactions involved in the control of flowering genes in the genome of Arabidopsis thaliana, a well suitable model organism for studying developmental regulation of gene expression and epigenetics. Among genes involved in flowering, our interest is focused on short vegetative phase (svp), a negative regulator of flowering. While the repressive effects of SVP on flowering are well studied, transcriptional regulation of SVP itself is still poorly understood. Therefore we aim to discover regulatory regions involved transcriptional regulation of SVP. Preliminary analysis of the locus via Chromosome Conformation Capture (3C), a technique that allows to monitor the formation of chromosomal interactions, indicates the importance of a region located about 2kb upstream of the SVP coding region. This region encompasses two CArG boxes and appears to be an interesting candidate for an SVP enhancer: i) CArG boxes are transcription factors (TF) binding sites for MADS-­‐box TFs, ii) SVP is a member of the MADS-­‐box TF family and iii) MADS-­‐box TFs are known to regulate the transcription of other family member. This internship will focus on testing the role of the SVP-­‐associated CArG-­‐boxes in the transcriptional regulation of SVP by using a transgenic approach. The student will make use of a construct containing the GFP reporter gene under the control of the full-­‐length SVP promoter region, and will generate a mutated version of the existing construct lacking the CArG-­‐
boxes. After stable integration of the two constructs into the Arabidopsis genome, the student will examine the effect of mutated CArG-­‐boxes on the GFP expression patterns and levels. This first transgenic approach will be complemented with a more challenging approach consisting in mutagenizing the endogenous candidate enhancer. This approach will rely on the recently reported genome-­‐editing tool CRISPR-­‐Cas. The student will be involved in generating Arabidopsis thaliana plants lacking the candidate CArG boxes. Techniques The project involves recombinant DNA technology, Arabidopsis transformation, DNA blot and PCR analysis, fluorescence microscopy. References o
o
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Fauser et al. (2014). The Plant Journal Posé et al. (2013). Nature Hövel I. et al. (2012) Methods, in Press. De Wit E. & de Laat W. (2012) Genes & Dev Gene regulation by epigenetics and chromosomal interactions
Master program:
Green Life Sciences
Group/Research institute: Nuclear organisation group/ Plant Dev & (epi)genetics;
Swammerdam Institute for Life Sciences
Contact person:
Dr. Maike Stam, [email protected], tel: 020-5257655
Supervisor:
Iris Hövel, [email protected]
The Stam subgroup studies the role of epigenetic mechanisms and chromosomal interactions in
gene regulation. Epigenetic gene regulation refers to changes in DNA methylation and histone
modifications that lead to changes in gene expression that are mitotically and/or meiotically
heritable. Chromosomal interactions refer to protein-mediated interactions between e.g. the
transcription start site (TSS) of a gene and cis-acting regulatory sequences. These regulatory
sequences can be nearby, in higher eukaryotes gene regulation, however, also involves specific
physical interactions between chromatin regions that are up to hundreds of kilo base pairs apart
from each other on the same chromosome or even on different chromosomes.
One of our aims is to identify long-distance interactions related to gene expression in the
genome of Arabidopsis thaliana, a well suitable model organism for studying developmental
regulation of gene expression and epigenetics. The chromatin organization in a nucleus is dynamic
and depends on the epigenetic state of the genome in each particular cell type. Thus, it is
important to restrict the analysis to a specific cell type to identify interactions related to gene
regulation. The analysis of a mix of different cell types would result in an overlay of different
chromosomal interaction patterns, hampering the identification and characterization of contacts
associated with the regulation of specific genes.
Isolating specific cell types from plant or animal tissue was laborious and tricky up to now.
A recently developed method allows the isolation of nuclei tagged in specific cell types (INTACT;
see video abstract at http://dx.doi.org/10.1016/j.devcel.2010.05.013). With this method, in the cell
type of choice, a fusion protein is
expressed that binds to the nucleus.
As a result, tagged nuclei of a
specific cell type can be isolated
from tissue that has a mixed cell type
composition. INTACT is based on
standard
laboratory
techniques,
cheaper and easier to accomplish
than comparable sorting methods
(e.g. FACS). In addition INTACT
allows the isolation of nuclei without
harsh treatments that may affect the
integrity of chromatin interactions.
The internship involves
establishing the INTACT method for Arabidopsis guard cells and phloem companion cells, a
specific cell type in leaf tissue. Arabidopsis plants were transformed with constructs containing cell
type-specific promotors and the coding region for the fusion protein. The transgenic lines will be
characterized by fluorescence microscopy, PCR and DNA blot analysis. Lines showing the
expected cell-type specific tagging of nuclei will be used to establish the INTACT method for guard
cells and phloem companion cells. INTACT will subsequently be used to perform cell-type specific
chromatin structure analyses.
Techniques
The project among others involves recombinant DNA technology, DNA blot and PCR analysis, fluorescence
microscopy, nuclei isolation, streptavidin pull-down (INTACT).
References
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Deal R.B. & Henikoff S. (2010) Dev Cell. 18(6): 1030–1040.
Steiner F. et al. (2012) Genome Res. 22(4): 766–777
Louwers M. et al. (2009) Plant Cell 21, 832.
Hövel I. et al. (2012) Methods, in Press.
De Wit E. & de Laat W. (2012) Genes & Dev. 26: 11-24
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.
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. We just discovered that three transcription factors on the pathogenicity
chromosome itself are involved in the expression of these effector genes. On the
pathogenicity chromosome are also six other transcription factors, and we would like
to answer the following questions:
- Can these six transcription factors promote or suppress effector gene
expression?
- Do the transcription factors affect virulence of the pathogen?
- Do the transcription factors have the same function in Fusarium strains with
different host plants?
A student who signs up for this challenge has already some great tools available:
- Fusarium with a reporter gene that shows (via GFP) when and where the
effector genes are induced.
- Fusarium overexpressing the transcription factors from the pathogencity
chromosome.
- Fusarium expressing RFP tagged versions the transcription factors.
- Genome wide expression data (RNAseq) from Fusarium infecting plants.
You can contact me for more details on the project plan (see below).
Techniques involved will be: plant infections, establishing novel infection assays,
microscopy, RNA isolation, qRT-PCR, Fusarium transformation.
This project is especially suited for a creative, independant student.
Group: Molecular Plant Pathology
Examiner: Martijn Rep, Supervisor: Lotje van der Does.
Interested? Questions? E-mail to: [email protected]
Localizing a toxin producing enzyme in a plant pathogenic fungus
Some fungi are able to infect plants, but we don’t fully understand how they do that.
In the tomato pathogen Fusarium oxysporum, several genes were identified that are
necessary for the fungus during infection. One of these genes is FOP1, which codes
for an unknown enzyme with a hydrolase domain. Without FOP1, Fusarium is no
longer pathogenic. A homolog of FOP1 in Alternaria (another plant pathogenic
fungus) is involved in the procuction of toxins. For Alternaria, toxins are necessary
for succesful infection.
Question
Both FOP1 and the homolog of FOP1 (from Alternaria) have a peroxisomal targeting
signal, suggesting that both enzymes perform their function in the peroxisome. In this
project, the aim is to test whether the FOP1 protein indeed localizes to the
peroxisomes.
Approach
- Transform Fusarium with RFP fused to a peroxisomal targeting signal. This
will stain the peroxisomes red.
- Transform the RFP expressing Fusarium with FOP1 fused to GFP. If FOP1
localizes to the peroxisomes, we should see colocalisation of RFP and GFP.
- The cloning (of RFP, FOP1, GFP) will be part of the project.
In addition:
- Test whether localization of FOP1 to the peroxisomes is important for its
function during infection.
Techniques involved
PCR, cloning, sequence analysis, microscopy and plant infections.
This project is especially suited for a systematic, independant student.
Group: Molecular Plant Pathology
Examiner: Martijn Rep, Supervisor: Lotje van der Does.
Interested? Questions? E-mail to: [email protected]
Manipulation of plant defenses: delivery of spider mite effectors by Pseudomonas Supervisor: Robert Schuurink, [email protected] MSc project (for at least six months) for Green Life Science students or other MSc students with a relevant background. Introduction
Some spider mites are known to manipulate the jasmonic acid (JA) and salicylic acid (SA)
mediated defenses in plants to their own benefit. It is thought that spider mites use proteins in
their saliva for this purpose that we call effectors. Through RNA-seq, bioinformatics and
proteomics we have identified putative salivary effectors of which several have been
characterized in detail. Using Agrobacterium tumefaciens mediated transient expression in
Nicotiana benthamiana we have shown that at least three effectors suppress increases in SA
levels caused by these bacteria themselves. We have also started using Pseudomonas syringae
pv tomato DC3000 (Pto) and pv tabaci (Pstab) to deliver the effectors to plants. Pto and Pstab
use the type III secretion system to deliver proteins to plants cells and several Pto mutants
exist with endogenous effectors deleted. This enables experiments in which only the spider
mite effector of choice is delivered to the plant. Using this system we have now indications
that there is a tobacco protein that specifically recognizes a particular spider mite effector.
Hypothesis and objectives
Spider mite effector Te153 is recognized in tobacco. The objectives are to test this hypothesis
and to identify tobacco proteins that interact with this effector.
Research and techniques
The student will be involved in measuring translocation of spider mite effectors by Pto and
Pstab and determining the phenotype of
the plant upon translocation. The
phenotype can be determined at various
levels: e.g. spider mite performance
assay, Pseudomonas competition assays,
hormone concentrations and defense
gene expression. The translocation will
be determined using a cAMP generating
reporter enzyme. cAMP levels can me
measured with a monoclonal anti-body
using an ELISA.
The student will also be involved the
identification of tobacco proteins that
interact with Te153 effector. To this end
several constructs have been and will be
made as such that the effector is tagged with a protein that is recognized by an antibody. This
tag will allow specific selection of this protein from a mix of proteins and to determine which
proteins bind to it. The latter will be done using an LCMS-based proteomics approach in
collaboration with the mass spectrometry department. Upon identification of the interacting
proteins, the most promising candidate(s) will be cloned and expressed (with a different tag)
to confirm the interaction. 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; GUS-­‐gene expression analysis; Arabidopsis Phenotyping -­‐ salt/drought tolerance; lipid 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.
Gene regulation by epigenetics and chromosomal interactions
Masterprogramma:
Green Life Sciences
Group/Research institute: Nuclear organisation group/Plant Dev & (epi)genetics; Swammerdam
Institute for Life Sciences
Contact person:
Dr. Maike Stam, [email protected], tel: 020-5257655
Supervisors:
Kathrin Lauss, [email protected]; Rechien Bader, [email protected].
The Stam subgroup studies the role of epigenetic mechanisms and
chromosomal interactions in gene regulation. Both levels of gene
control are essential for normal growth and development.
Epigenetic gene regulation refers to mitotically or meiotically
heritable changes in gene expression due to changes in DNA
methylation and chromatin structure; The DNA sequence stays the
same. The maize plants in the figure e.g. have the same DNA, yet
display another color. Chromosomal interactions refer to proteinmediated interactions between the transcription start site (TSS) of a gene and its regulatory
sequences. Intriguingly, besides gene control by nearby cis-acting regulatory elements, gene
regulation in higher eukaryotes also involves physical interactions between chromosomal regions
up to hundreds of kilobase pairs apart on the same chromosome (cis), or even between regions
located on different chromosomes (trans).
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 and chromatin structure. B’ is
low expressed (light pigmented plants), while B-I is high expressed (dark pigmented plants). 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 can enhance the transcription of the b1 gene. 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
mendalian rules, is called paramutation. The regulatory sequences 100 kb upstream of the b1
coding region are required for the in trans inactivation of B-I. Recent data indicates that the
molecular mechanisms underlying the in trans inactivation require the small RNA-directed DNA
methylation (RdDM) pathway.
The internship involves an exciting project in which advanced molecular biological techniques will
be used to study the molecular mechanisms underlying paramutation. More specifically, the
internship will focus on the mechanisms underlying the in trans silencing, To allow studying the
underlying mechanisms, first the time point at which the paramutation process starts will be
determined by analyzing the transfer of DNA methylation from B’ to B-I. Previous experiments
showed that, when crossed together, the B-I regulatory sequences become methylated early in
plant development and we are in the process of defining the exact time point at which the DNA
methylation process starts. Once defined, we will also examine to potential role of small RNAs in
this process.
Techniques
The project amongst others involves recombinant DNA technology, DNA methylation analysis (bisulfite
sequencing), siRNA analyses, (q)PCR and working with maize.
References
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Chandler V.L., Stam M. (2004) Nat Rev Genet. 5, 532-44.
Stam M., Mittelsten Scheid O. (2005) TIPS 10, 283-290.
Stam M. (2009) Molecular Plant 2009 2(4):578-588; doi:10.1093/mp/ssp020.
Louwers M et al. (2009) Plant Cell, 21, 832.
Haring M et al. (2010) The Plant Journal, 63, 366-378.
Belele et al. (2013) PLoS Genetics, In Press.
Virulence of the melon pathogen Fusarium oxysporum f. sp. Melonis Supervisor: Sarah Schmidt, [email protected]; alternative contact Martijn Rep Fusarium oxysporum (Fo) is a soil-­‐borne, pathogenic fungus that infects plants through the roots, growing in the vascular system of the plant and causing Fusarium wilt disease. It is a species complex consisting of various formae speciales. Each forma specialis is able to cause disease on a particular host plant among them several important crop plants such as tomato, melon, cotton, basil, pea and banana. In this project you will be working with the Fo strains infecting melon and tomato plants. To be able to infect a host plant, the fungus secretes small proteins in the host xylem sap to manipulate the host and to create a favorable environment for the fungal growth. These small proteins are called effectors or SIX (Secreted In Xylem). Via transcriptomics we have identified a family of fungal-­‐specific transcription factors that seem to regulate the effector genes. To test whether these regulators are required for fungal pathogenicity, you will make gene knock-­‐out mutants of these regulators and test them in a bioassay with tomato plants. Techniques: PCR, fluorescent microscopy, creating gene knock-­‐outs, and mutant strains, Agrobacterium-­‐mediated fungal transformation, molecular cloning, greenhouse disease assays Requirements: smart, independent, motivated, knowledge of molecular lab techniques Functional analysis of virulence genes in the plant pathogen Fusarium oxysporum
Fusarium oxysporum is a pathogenic fungus that resides in the soil and is able to infect a
large variety of plants, causing wilt disease. In this project, we sequenced the genomes of
several strains of F. oxysporum that are pathogenic to cucurbits (cucumber, melon,
watermelon). Cucumber-pathogenic strains do not cause disease on other host plants than
cucumber, and this host specificity is also found in the interactions of melon- and
watermelon-infecting F. oxysporum.
We think there must be a genetic basis underlying this narrow host range of each of these
strains. Evolutionary selection pressure causes the genes responsible for infection
(virulence or ‘effector’ genes) to mutate at a high rate.
Using comparative genomics, we try to get an idea of the effectors necessary for each of
these hosts and also to see whether a set of effectors can be found that are ‘cucurbitspecific’.
You will investigate the genetic diversity in effector genes by comparing cucurbit-infecting
strains of F. oxysporum, in various ways:
-­‐
Screening for presence and expression of known effector homologs and possible
(predicted) virulence genes
-­‐
Functional analysis of these genes (knock-out/complementation) to evaluate their
relevance and function during plant infection.
-­‐
Pathogenicity bio-assays in the greenhouse
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),
molecular cloning, genetic modification of fungi, DNA/RNA isolation, comparative
genomics, (RT-)PCR, microbiological lab techniques, fluorescence microscopy.
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].
Role of PLC in drought-­ and heat-­stress tolerance of 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; 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 will be the main study of our proposal. In India, the research will be focussed on rice while in the
Netherlands, Arabidopsis will be the main plant of interest. The research is ment to explore the potential
of PI metabolites, 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. Aug 22. pii: pcu116. [Epub ahead of print]. The metabolic connection between color and scent in Petunia Supervisor: Nur Fariza M. Shaipulah, Plant Physiology, SILS, University of Amsterdam. Email: N.F.B.M-­‐[email protected]. Alternative contact: Robert Schuurink Petunia is known for its variation in flower color, size and scent production. Color and scent metabolites are derived from L-­‐Phenylalanine via the phenypropanoid pathway. Two R2R3 MYB transcription factor, ANTHOCYANIN 2 (AN2) and ODORANT1 (ODO1) regulate the anthocyanin and volatile phenypropanoid/benzenoid biosynthesis respectively (Quattrocchio et al. 1999; Verdonk et al. 2005). Mutation in AN2 coding region leads to less-­‐pigmented flowers, and ODO1 silencing results in a severe reduction of phenypropanoid/benzenoid volatiles. We have recently isolated three ODO1-­‐dependent biosynthetic genes; Caffeoyl CoA O-­‐
Methyltransferase (CCoAOMT) from Petunia hybrida cv. Mitchell petals. Mitchell has white flowers and emits 14 volatile phenylpropanoid/benzenoids. Knockdown of CCoAOMT1 did not only affect the amount of volatiles, but also lead to the accumulation of anthocyanin in plants. The transgenic plants have purple leaves and stems, as well as pink-­‐blush flowers (Figure 1). The question is how the color production is activated in these transgenic lines. To address the functions of CCoAOMT in phenypropanoid/benzenoid pathway, we made a hairpin construct of CCoAOMT2 and CCoAOMT3 for stable transgenic lines. With these lines we will investigate the roles of CCoAOMT2 and CCoAOMT3 in the phenypropanoid/benzenoid pathway. Next, the transgenic plants of PhCCoAOMT2/3 will be crossed to PhCCoAOMT1 silenced lines to examine the effects on volatiles, anthocyanin and lignin –also produced via the phenylpropanoid pathway -­‐ biosynthesis in Petunia. Students will use various techniques; RNA isolation, quantitative Reverse Transcriptase-­‐
PCR (qRT-­‐PCR), volatile trapping and extraction, Gas Chromatography-­‐Mass Spectra (GC-­‐MS) analysis and plant transformation. Objectives: 1) To examine the expression profile of CCoAOMT2 and CCoAOMT3. 2) To identify knockdown lines of CCoAOMT2 and CCoAOMT3 and their phenotype 3) To analyze internal volatiles and emission of silenced lines compared with wild type. 4) To quantify the internal pools of CoA ester from silenced lines. Figure 1: Mitchell (WT) produces white (opening) flowers, meanwhile CCoAOMT1 silenced line shows pink-­‐
blush flowers (line 4). Line 4 WT Quattrocchio F, Wing J, van der Woude K, Souer E, de Vetten N, Mol J, Koes R (1999) Molecular analysis of the anthocyanin2 gene of petunia and its role in the evolution of flower color. Plant Cell 11 (8):1433-­‐1444 Verdonk JC, Haring MA, van Tunen AJ, Schuurink RC (2005) ODORANT1 regulates fragrance biosynthesis in petunia flowers. Plant Cell 17 (5):1612-­‐1624. doi:10.1105/tpc.104.028837 Free University, Francesca Quattrochio; [email protected] The vacuole of plant cells is an organelle with a large variety of crucial functions for the organism life. Although this compartment is seen as the homologue of the animal cells lysosome, its function appears to be more complex. It is involved in sequestration and storage of a variety of different molecules and is the host space of enzymatic reactions that require specific conditions, but it also defines the turgor of the cell, it represents a constant buffer of ions and other compounds necessary to survive when the environment makes sudden changes, it is dynamically connected with the the endomembrane system of the cell to allow transport of cargos from and to the outside, and probably it has several more functions that we still do not know. Specialization of vacuoles also occurs, so that in a single cell it is often possible to find several vacuoles with different functions (Frigerio et al., 2008). In spite of this crucial role of the vacuole for plant life, still very many aspects of its genesis and physiology are unknown. In Petunia hybrida several loci are known to be required for the activation of anthocyanin pigments biosynthesis, their accumulation and stabilization in the vacuole and the acidification of the vacuolar lumen necessary for the display of the color (Koes et al., 2005). The PH1-­‐PH7 loci result, if mutated, in a change of petal color from red to blue, due to the shift of anthocyanin absorption spectrum in response to an increased vacuolar pH, while the ANTHOCYANIN 1 (AN1) and ANTHOCYANIN11 (AN11) loci affect next to vacuolar acidification, also anthocyanin biosynthesis (Koes et al., 2005). Some of these genes encode regulatory proteins such as, AN1 (PH6, proved to be the same then AN1) and AN11 which are bHLH and a WDR regulators of transcription (de Vetten et al., 1997; Spelt et al., 2002)), the transcription factors PH3 ((Verweij, 2007), and Verweij, Koes, Quattrocchio; submitted) and PH4, the latter functioning in one complex with AN1 (Quattrocchio et al., 2006a). Other of these genes encode structural proteins: PH5 codes for a P-­‐type H+-­‐ATPase localized on the tonoplast (Verweij, 2007) and PH1 another P-­‐ATPase which interacts with PH5 to form an active pump able to produced a strong proton gradient in vacuolar lumen against the electrochemical gradient. The combined expression of these two pumps can restore the pH phenotype in any of the regulatory mutants, but other defects proper of an1,an11, ph3 and ph4 mutants are not rescued in this way. The analysis of ph3, ph4 and an1 mutants had revealed that next to the proton homeostasis in the vacuolar lumen, there is much more going wrong in the mutant cells. One process affected in an1, ph3 and ph4 mutants, is the stability of anthocyanin molecules. Disappearance of petal color after opening of the bud (flower color fading) has been observed when the ph3, ph4 or ph6 (an1G621, an allele of AN1 that affects vacuolar acidification without blocking pigment production and accumulation) mutants are in a genetic background containing the dominant FADING (Fa+) allele, whereas no fading takes place in ph1 or ph5 mutants occurring in Fa+ lines (Quattrocchio et al., 2006a). Thus fading is not just consequence of the increased vacuolar pH, but rather of additional vacuolar defects in ph3, ph4 and an1 (see Figure1).We have recently isolated the FA locus by transposon tagging and we are now in ther process of characterizing its function. By RNA-­‐profiling we identified some 10 genes, which expression is strongly reduced in an1, ph3 and ph4 mutants (Verweij, 2007). Two of these turned out to be PH5 and PH1, while the other differentials are possibly encoding factors required for vacuolar genesis or vacuolar content stability and might be responsible for the absence of vacuolinos in the an1, ph3 and ph4 mutants and for fading. Thus the mechanism underlying the dramatic color change observed in fading petunia mutants (Figure 1) requires further investigation. Fig. 1: phenotype of the same flowers photographed on the day it opened the corolla, and each subsequent day. The petunia line carries the Fa+ locus and an unstable mutation at the PH4 gene. The red spots (revertant for PH4) do not fade. (from Quattrocchio, 2006 #1573). Purpose of the proposed research The purpose of this study is to understand how the stability of vacuolar content is determined via de study of specific mutants that affect this characteristic. The unraveling of the mechanisms at the basis of flower color fading will contribute to better understand vacuolar physiology and to develop tools to improve vacuolar stable accumulation of secondary metabolites. This is required for all biotechnological strategies that see plant cells as “green factories” that produce large amounts of valuable compounds. Many of these compounds find applications in medicine (e.g. cytostatic, morphine and the newly developed anti EBOLA drugs) , in human diet (antioxidants, flavors), in animal feeding (tannins) Fig2. New unstable fading mutant . The revertant spots shown in the small inserts represent reversions to the fading phenotype. Figure 3. ph4L2104 and its target genes. Techniques that will be used during this internship Analysis of segregating populations by Phenotype and genotype DNA/RNA isolation PCR Real time analysis of gene expression Isolation and analysis of anthocyanins DNA sequencing In vitro cultivation of plant material Generation of transgenic plants Microscopy (light and confocal)