Thesis projects in the Phytopathology lab Galacturonic Acid

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               Thesis projects in the
                Phytopathology lab
                   (2009/2010)


     Phytophthora
       infestans
                                                                   C
                                                                   f
                               Molecular
                              Ecology and
                                                  AVR
                               Biocontrol
                                                    Cladosporium




                                                        Solanaceae
                                                        defense
Verticillium
                                Botrytis
                                cinerea




                    Website: www.php.wur.nl/UK/
                           The Cladosporium –group
                             (contact:pierre.dewit@wur.nl)
The biotrophic fungus Cladosporium fulvum (syn. Passalora fulva) is the causal agent of leaf mold of
tomato (Lycopersicon ssp). The interaction between C. fulvum and tomato complies with the gene-for-
gene model (Figure).




The gene-for-gene interaction between C. fulvum and tomato. Matching combinations of a pathogen
avirulence gene (Avr) and a plant resistance gene (Cf) result in a hypersensitive response (HR).

We are very interested in understanding the communication between the host plant and the pathogen
through Avrs also called effectors. The group performs bioinformatics, biochemical, molecular and cell
biological studies in which MSc students can participate.

1. The interactions between the Avrs and Cf proteins. C. fulvum Avr2 binds to tomato protease Rcr3
and initiates a Cf-2-dependent hypersensitive response. Interaction between Avr2 and Cf-2 also
requires Rcr3, a cysteine protease. Various molecular techniques will be used to confirm interaction
between the three proteins (guodong.Wang@wur.nl )

2. Functional analysis of homologous effectors occurring in fungi related to C. fulvum. So far
homologues of the C. fulvum effector proteins had not been identified in other fungal species. Now the
genome of C. fulvum is available which allows comparative genomics with effectors occurring in other
sequenced fungal species (ioannis.stergiopoulos@wur.nl).

3. Identification of novel effectors in the genome of C. fulvum. By proteomic and bio-informatic
analyses novel secreted effectors of C. fulvum can be identified. The newly discovered effectors will
be studied in more detail by other members of the group (harrold.vandenburg@wur.nl).

4. Functional analysis of novel effectors of C. fulvum. Putative novel effectors need to be analyzed in
vivo. Their effect on activating and or suppressing of defense responses will be analyzed.
(rahim.mehrabi@wur.nl); (bilal.okmen@wur.nl); (mansoor.karimi@wur.nl).

Further information can be found on the website: http://www.php.wur.nl/UK/Research/Cladosporium.
         Molecular Ecology and Biological Control
                           Contact: jos.raaijmakers@wur.nl
The overall goal of the research program of the Molecular Ecology Group is to
understand the ecological, biochemical and molecular mechanisms involved in
interactions between beneficial bacteria, plant pathogens and plants. The program
focuses on the ecology, evolution, genomics and metabolomics of plant-associated
bacteria and in particular on beneficial Pseudomonas species.




MSc research topics
1. Plant Growth Promotion and Plant-Bacteria Interactomics
Beneficial bacteria can directly promote plant growth, alter root architecture and
induce systemic resistance in plants against pathogens. The underlying mechanisms
are being studied using a variety of techniques and approaches.
Contact: judith.vandemortel@wur.nl

2. Biological control of Pathogenic Fungi and Oomycetes
When applied to seeds or planting material, beneficial bacteria offer an attractive
alternative to currently used disease control strategies. The overall aim is to identify
and characterize antagonistic bacteria from diverse agro-ecosystems (Vietnam,
Ghana) and to exploit their potential to control important plant pathogens.
Contacts: cuong.le@wur.nl; andrews.akrofi@wur.nl

3. Role of Beneficial Bacteria in Natural Disease-Suppressive Soils
Specific soils have been identified worldwide in which beneficial microorganisms
protect plants against infections by soil-borne pathogens. The microorganisms and
mechanisms involved in this natural disease suppressiveness are largely unknown
and will be investigated by microbiological and genomics-based approaches.
Contact: rodrigo.mendes@wur.nl

4. Genomics and Metabolomics of Beneficial Bacteria
Genomics and biochemical approaches are used to identify novel bacterial genes,
traits and bioactive compounds that play an essential role in the behaviour and
activities of plant-associated bacteria, including motility, biofilm formation, root
colonization, survival and defense against predators and competitors.
Contacts: chunxu.song@wur.nl; jos.raaijmakers@wur.nl

More information:
Web: http://www.php.wur.nl/UK/Research/MolecularEcologyandBiologicalControl/
E-mail: jos.raaijmakers@wur.nl
                                        The Phytophthora group
                                              contact: francine.govers@wur.nl
The overall goal of the Phytophthora group is to unravel the mechanisms underlying
pathogenicity in plant pathogenic oomycetes, in particular in the potato late blight pathogen
Phytophthora infestans, one of the most devastating potato pathogens worldwide and difficult
to control. The ongoing research is centered around two main topics (i) Phytophthora-host
interactions and (ii) target discovery towards novel oomicides.

                                                                         resistent   P. infestans population susceptible
     Phytophthora
                                                                                                            Avr1
                                                                                                           deletion




                                                                                                          Avr3a
                                                                                                        two alleles



                          haustorium
                                                                                                           Avr4
                                                                                                        ‘frameshift’



             plant cell
                                                                                                        Avr-blb1

                                       RxLR                                                              Multiple
                                                                                                         variants




Phytophthora RxLR effectors: keys to durable late blight resistance
Phytophthora produces numerous effectors, many of which are transported to the inside of
the cell where they suppress defence responses and manipulate the host cell machinery.
Recognition of effectors by their cognate resistance (R) proteins results in a hypersensitive
response that blocks further growth of the pathogen. Breeders have introgressed late blight
R genes into cultivated potato but in the field this resistance is quickly defeated. P. infestans
can easily mutate its effector genes thereby avoiding recognition. Insight into the molecular
interaction between effector and R protein can help in designing more rational breeding
strategies. The hallmarks of oomycete effectors are an N-terminal signal peptide followed by
a cell-targeting motif (RxLR), and a C terminal domain of variable length that is highly diverse
and has no homology to any known protein.
      MSc research projects offered within this theme are:
      - functional analysis of RxLR effector genes by in planta expression using
        Agrobacterium tumefaciens transient assays (ATTA) and domain swapping to
        determine which part of the C-terminal domain is responsible for effector activity
      - identification of novel effector targets by yeast-2-hybrid screening
      - functional analysis of effector targets by VIGS (virus induced gene silencing).

Novel anti-Phytophthora agents: a rational strategy for target discovery
Genome mining in Phytophthora has resulted in an avalanche of information that can be
exploited to search for unique features of oomycetes. In our mining expeditions focused on
phospholipid modifying enzymes and G-protein signaling components, we found many genes
encoding novel proteins not found in any other organism. Novel genes are intriguing. Are
they key to potential anti-oomycete agents?
     MSc research projects offered in this theme involve studies on two novel classes of
     proteins: secreted phospholipase D’s that could play a role in degradation of host
     membranes and transmembrane receptors named GPCR-PIPKs. Activities may include
     cloning, expression analysis, biochemical assays, bioinformatics, in planta expression
     assays, in situ localization using microscopy, mutant analysis or expression in
     heterologous hosts.

Wesites: http://www.php.wur.nl/UK/Research/Phytophthora/ and http://www.php.wur.nl/UK/Publications/Phytophthora/
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Research projects on Botrytis

Contact: jan.vankan@wur.nl



Botrytis cinerea (grey mould) causes devastating pre- and post- harvest diseases in
more than 200 plant species, including economically important crops (grape, tomato,
cucumber, strawberry, rose, cut flowers, potplants). B. cinerea produces and secretes
a spectrum of phytotoxic metabolites and hydrolytic enzymes. Our research aims to
understand which factors enable B. cinerea to cause disease and which plant factors
contribute to (partial) disease resistance.

Project 1

Galacturonic acid metabolism in Botrytis cinerea
Pectin is an important component of plant cell walls. Pectin is a complex polymer
largely composed of the sugar galacturonic acid. B. cinerea preferentially grows on
plant tissues that are rich in pectin, and the production of pectinases is important for B.
cinerea to cause disease. All evidence from prior research suggests that pectin is the
favorite food for B. cinerea. We study genes involved in galacturonic acid metabolism
and their importance for virulence.


Project 2

Genetic identification of Botrytis resistance loci in Arabidopsis
B. cinerea causes plant tissue rot by secreting a spectrum of pectinases. Infiltration of
purified pectinases into plant leaves causes cell wall hydrolysis, tissue collapse and
the occurrence of necrosis. Using a genetic screen, we identified a recessive locus in
Arabidopsis that confers resistance to damage caused by infiltration with pectinases.
The aim of the project is to clone and characterize the gene(s) that confers resistance
to B. cinerea pectinases and unravel the resistance mechanism. We hypothesize that
plants with resistance to B. cinerea pectinases show (partial) resistance to B. cinerea.


Project 3

Gene expression in Botrytis cinerea during sexual development
Botrytis cinerea is capable of sexual development in the field. The fruiting body (called
‘apothecium’) can also be produced in the laboratory (see image below). Little is
known about fungal gene expression in the apothecia and about the genes required
for apothecium development. Gene expression in apothecia at different stages of
development will be studied using microarray analysis and RT-PCR. The function of
selected genes in apothecium development will be studied using targeted mutants.
                                                                SOL group
                                           Innate immunity in Solanaceae
In the Solanaceae (SOL) group of the Laboratory of Phytopathology (Plant Sciences Group) we want to unravel how plants are able
to resist harmful pathogens. We focus on Solanaceous plants as these represent economically important crops, such as tomato and
potato, and they are also versatile model plants in the lab. Our main interest is to exploit resistance traits that are present in nature
to generate pathogen-resistant plants. With such plants, the use of harmful chemicals in food production can be diminished. We aim
to sort out, using a very diverse set of techniques, how pathogens are perceived by resistant plants, how resistance (R) proteins
trigger defence and which downstream signalling protein cascades are activated. Furthermore, we study how successful pathogens
can manipulate these responses and suppress them.

                                                             Ongoing projects
Unravelling the nuclear function and import                                          Functional analysis of phospholipid signalling genes
mechanism of the plant immune receptor Rx                                            to investigate their role in plant disease resistance
Rx from potato mediates extreme resistance to Potato Virus X                         This line of research     1
(PVX). Due to the lack of a signal peptide, this resistance protein                  focuses on the enzymes
was predicted to localize to the cytoplasm. However, it has been                     that are responsible for
discovered that it localizes not only to the cytoplasm, but also to                  the rapid modification of
the nucleus. Similar immune receptors have now also been found                       specific phospholipid
in the nucleus. Our research aims to understand the role that                        signalling molecules in
immune receptors play in the nucleus and how they can be                             tomato and Nicotiana
imported there without carrying a nuclear targeting signal.                          benthamiana upon
                                                                                     pathogen challenge.
                                                                                                       2   A                                      B
                                                                                                                                                       DAG

                                                                                                           kDa       Empty vector PLC-GST
                                                                                                           100
                                                                                                            75                              PLC-GST

                                                                                                           25                               GST tag




                                                                                                                                                      PI

                                                                                    1 - Phospholipid signalling upon Avr4 recognition by Cf4           time


                                                                                    2 - Purification of a recombinant phospholipase C (PLC) protein from E.coli
A DNA construct expressing the N-terminal domain of Rx fused to GFP was             and assay of its enzymatic activity. A- SDS-PAGE protein analysis shows the
transfected to Arabidopsis protoplasts, which were subsequently subjected to        purification of a plant PLC protein fused to a GST tag. B- purified GST-PLC
confocal microscopy. The GFP-fusion localizes to both the cytoplasm and nucleus.    hydrolizes phosphatidyl-inositol (PI) and thereby produces diacylglycerol (DAG).


Characterization of NRC1, a key protein required for                                 How do extracellular immune receptors detect
defence signalling downstream of multiple resistance                                 virulence proteins from C. fulvum and subsequently
proteins                                                                             activate the defence response?
NRC1 expression is up-regulated upon infection with the fungus C.                    We focus on elucidating the perception mechanisms of
fulvum. Currently we are studying the composition of the NRC1                        virulence proteins such as Avr4, by extracellular receptors.
complex in order to understand the signal transduction pathway
triggered by NRC1, leading to a defence response.

                                                                                                                     susceptible
                                            Silencing of NRC1 in N. benthamiana
                                            affects the defence response induced
                                            by several R proteins. N. benthamiana
                                            plants were silenced for NRC1
                                            (TRV:NRC1) or not (TRV:00). Three                                            resistant
                                            weeks later, the defence response
                                                                                  This cartoon shows that a plant with immune-receptor Cf-4 (glasses) can see and
                                            (programmed cell death) was activated
                                                                                  resist the attacker with Avr4 (fork), whereas a plant without Cf-4 cannot. On the
                                            via several R proteins.
                                                                                  right the responses to Avr4 in a susceptible and a resistant plant are shown.

As a student you can join any of these projects. We work with a very diverse toolbox. You can work with DNA and clone genes,
isolate RNA, perform PCRs, study protein-protein interactions in yeast and in plants and produce proteins in bacteria. You can
perform confocal microscopy, cell death assays and test extreme virus resistance, silence genes in plants, and study how
phospholipids trigger resistance mechanisms of plants against pathogens.
When interested, contact Matthieu Joosten (matthieu.joosten@wur.nl, 0317-483411). For more information also visit our website:
http://www.php.wur.nl/UK/Research/SOLgroup/

				
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Description: Thesis projects in the Phytopathology lab Galacturonic Acid