Research interest

I am interested in the molecular mechanisms of gene-for-gene based resistance in plants and strategies employed by pathogens to overcome disease resistance. Resistance (R) proteins recognize specific pathogens and trigger activation of signal transduction cascades that induce defense responses that restrict pathogen ingress. My major research questions are: A) how does a plant recognize a pathogen, B) how is defense signaling activated and what is the role of post-translation modifications in this process and C) how does a pathogen avoid or suppress defense responses?

Our main model is the interaction between tomato and the soil-borne fungal pathogen Fusarium oxysporum fsp. lycopersici (see figure 1). Fusarium colonizes the xylem vessels of a plant causing wilt disease. During infectionthe fungus secretes many small proteinsinthe xylem sap that are referred to as SIX protein (secreted in xylem). In our current projects we are studying the role of these proteins for the fungus and the role they have in the infection process. For instance Six3 (Avr2) was found to be required for full virulence on susceptible plants. Currently we are investigating the ability of SIX proteins to interfere with the induction of host defense.

Besides virulence factors some of the SIX proteins are unintentionally recognized by plant R proteins resulting in the activation of host defenses. One example is Avr2 that is recognized by the tomato I-2 resistance protein. Having the complete gene-for-gene pair in hand allows us to address the first research question: How does a plant recognize a pathogen? Most R proteins, including I-2, belong to the large family of NBS-LRR proteins. These proteins contain a central nucleotide-binding (NB) domain, an N-terminal coiled-coil domain and a C-terminal Leucine Rich Repeat (LRR) domain. The LRR together with the N-terminus is thought to provide recognition specificity, whereas the central NB domain acts as a regulatory switch and is predicted to regulate downstream signaling (for its predicted structure see figure 2). To understand how R proteins are activated we perform biochemical analysis of the nucleotide binding properties of wildtype and mutant proteins and we have analyzed the intramolecular interactions of the different domains. Based on these data we proposed a working model for these proteins in which the nucleotide binding state controls their activity (On/Off state) (Figure 3). This model was recently published in an invited review in Science (Takken and Tameling, 2009). To understand how an  R protein activates downstream signaling we use them as baits to identify interacting partners.

Furthermore, I am interested in the role SUMO has in defence signalling. This interest was raised after linking the observationsthat 1) some plant pathogens produce SUMOproteases that enhance their virulence and 2) plants affected in SUMO ligation ( siz1-2 mutant) show a constitutive defence phenotype. SUMOylation of proteins frequently results in recruitment of chromatin-modifying enzymes to transcription sites and thereby indirectly regulates gene expression. We presented a working model in Trends in Plant Science (van den Burg and Takken, 2010) in which we propose that SUMO modifying the epigenetic code of many defence genes. Our recent work (published in Plant Cell, van den Burg 2010 ) revealed that SUMO affects salicylic acid-dependent gene expression and acts as a translational repressor. We are currently investigating how this repression is released upon pathogen attack resulting in the induction of defence gene expression.

• To read more about my work click here.

Working model for the molecular arms-race between tomato and Fusarium.

a) Non-pathogenic Fusarium oxysporum strains trigger basal defenses preventing disease. Recognition is likely conferred by receptor-like kinases (RLKs) recognizing fungal PAMPs -triggered immunity (PTI) b) Effectors, suchas Avr2, suppress these responsesallowing disease. c) Recognition of Avr2 by the NB-LRR protein I-2 triggers its conformational change resulting in the activation of host defense. d) I-2 mediated defense can be blocked by Avr1, resulting in disease development. e) Plant evolved the I resistance protein to allow recognition of Avr1 and subsequent activation of host defense.
(Figure adapted from Takken and Rep (2010) MPP, 11, 309-314)

3D structure of the NB-ARC domain

Computationally derived 3D structure model of the NB-ARC domain of the resistance protein I-2. The model was created using the ADP bound structure of human APAF-1 (PDB code 1z6t, chain A) as  template. Locations of R protein specific motifs are marked with arrows. Amino acids of the MHD motif as well as the sensor I arginine are shown in stick representation. ADP atoms are depicted as balls-and-sticks. Subdomain coloring is: NB: red, ARC1: green, ARC2: blue. Atom coloring is: oxygen: red, nitrogen: blue, phosphorus: orange.
(Figure adapted from van Ooijen et al, (2008) J. Exp. Bot, 59 (6):1383-1397)

Model for the switch function of NBS-LRR proteins.

  In the absence of a pathogen an NB-LRR protein resides in its resting (ADP) state, in which the LRR stabilizes the closed conformation. The recognition platform forthe AVR protein  is provided by the C-terminal part of the LRR together with CC/TIR domain (CC). Perception of the AVR (direct or indirect) changes the interaction surface between the N-terminal part of the LRR and the ARC2subdomain, thereby releasing the autoinhibition conferred by the LRR.Subsequent nucleotide exchange triggersasecondconformational change, altering the interactions of the NB-ARC domain with CC and LRR domains (induced state). In the activated state the NB subdomain is accessible to interact with downstream signalling partners. Hydrolysis of ATP could return the protein to its resting state. (figure adapted from Takken and Tameling (2009) Science, 324;744-746)

Plant pathology group

The Plant Pathology group is headed by Prof.dr. Ben Cornelissen. The group is divided into two subgroups: my "plant" group and the "fungus" group headed by dr. Martijn Rep. In close collaboration both sites of the interaction between plants and their fungal pathogens is being studied. 

• Molecular Plant Pathology Group-UvA

• Martijn Rep

• Harrold van den Burg

People involved:

Xiaotang Di
    PhD student: The Avr2 effector protein from Fusarium as a guide to unravel plant innate immunity.
Lingxue Cao
    PhD student: Elucidating the molecular mechanism underlying the virulence and avirulence functions of the Fusarium Avr2/Six5 effector pair.
ir. Lisong Ma
    PhD student: Analysing the intrinsic functions of Avr2 and its perceival by the resistance protein I-2.     
Fleur Gawehns
   PhD student: Functional characterisation of Fusarium secreted effector proteins in disease and resistance

Dr Hanna Richter
  Technician: involved in all ongoing projects in the group.
Former lab members
ir. Ewa Lukasik
     PhD student: focussing on the ATPase activity of the NBS domain of R proteins and the identification of interacting protein
drs Vladimir Krasikov
    Postdoc analyzing the role of the Xsp10 protein for resistance anddisease to Fusarium oxysporum
dr. ir. Harrold van den Burg
    Veni Postdoc studying the role of SUMO modification in plant defence signalling
ing Marianne de Vroomen
   Technician, involved in most ongoing projects.
Drs Mobien Kasiem
  PhD student: PhD student: Structure and function of tomato disease resistance proteins
Dr Gerben van Ooijen
  PhD student: Structure and function of tomato disease resistance proteins
Dr Klaas Jan de Vries
  Postdoc:  Targeted proteomics of signalosomes mediating pathogen resistance
Dr Wladimir Tameling
  PhD student: Disease resistance proteins of the NBS-LRR class, molecular switches of plant defence
Dr Sergio de la Fuente van Bentem
   PhD student: Unfolding plant disease resistance, the involvement of HSP90 and its co-chaperone  PP5 in I-2 mediated signalling
Dr Jack Vossen
  Postdoc: Characterisation of the I-2 signalosome
Dr Sandra Elzinga
   Postdoc: Characterisation of the Mi-1 signalosome

• Link- uva

• Molecular Plant Pathology group

• Swammerdam Insitute for Life Sciences

Invited speaker

Selection from the last five years

• The role of Fusarium effectors in NLR-mediated innate immunit.y XV International Congress on Molecular Plant-Microbe Interactions (IS-MPMI) July 29-August 2, 2012, Kyoto, Japan
  
• How to build a pathogen detector; structural basis of NB-LRR function. EMBO practical course 18-29 june 2012 TSL, Norwich, UK
• The role of Fusarium effectors in NLR-mediated plant innate immunity. a9 April 2012, Plant Biology Department, University of Fribourg, Fribourg Switzerland.
• The role of pathogen effectors in NLR-mediated plant innate immunity. International Meeting of the Collaborative Research Center SFB 648  "Communication in Plants and their Responsesto the Environment" May 19-22, 2011, Halle (Saale), Germany 
• Molecular co-evolution in the tomato-Fusarium pathosystem. October 8, 2010, Justus Liebig University , Giessen, Germany
  
• Functional analysis of Fusarium oxysporum f.sp. lycopersici effector proteins. DFG funded colloquium: 'Microbial reprogramming of plant cell development' priority project SPP1212 'Plant-Micro' June 2010,  Freising, Germany
• The arms race between effectors of Fusarium oxysporum and resistance proteins of tomato. Seminar series School of Biological and Biomedical Sciences, May 2010. Durham University, Durham, UK
  
• How to resist a resistance protein? British Society for Plant Pathology, Presidential Meeting, 2009, Oxford, UK
• How to resist tomato resistance proteins? XIV International Congress on Molecular Plant-Microbe Interactions. July 19-23, 2009, Quebec, Canada.
• Resistance proteins: scouts of the plant innateimmune system. 18th EUCARPIA congress, "Modern Variety Breeding for Present and Future Needs" 9-12 September 2008, Valencia, Spain
• Structure-function analysis of plant NB-LRR disease resistance proteins. SFB 670 " Structure Function and Evolution of Innate immunity" 22-23 September 2008, Max-Planck-Institut für Züchtungsforschung, Koln, Germany
• Resistance proteins: scouts of the plant innate immune system. EPS/Bioexploit Summerschool "On the evolution of plant-pathogen interactions: from principles to practices" 18-20 June 2008, Wageningen, the Netherlands.
• Resistance proteins: scouts of the plant innate immune system.  4th EPSO Conference, 22-26 June 2008 , Presqu'ile de Giens (Côte d'Azur), France
•  Molecular aspects of I-2 mediated resistanceto Fusarium oxysporum . Seminar series. May 2008, Max-Planck-Institut für Züchtungsforschung, Koln, Germany
• Function of the Nucleotide Binding Domain for RProteins. Keystone Symposium on Plant Innate Immunity, Feb 2008 Keystone Resort,  Keystone, Colorado, USA
• The NB-ARC domain of R proteins acts asa molecular switch regulating plant innate immunity.  (2007) Max von Pettenkofer Institut, LMU München, Germany
• Host Pathogen interactions from Plants to Mammals - Distinct and shared pathways of immune defense. Inflammatory Diseases of Barrier Organs 2007-Genetic exploration leadsto novel therapies in inflammation, Kiel, Germany.
• The NB-ARC domain: an NTP-hydrolysing molecular switch. Banbury meeting: Comparative Biology of Innate Immune Systems, (2006) New York, USA
• Innate immunereceptors in plants and animals; do analogous modules have comparable functions? EPS Symposium Intracellular signalling, (2006)Amsterdam, UvA
• R proteins, molecular switches of plant defence, 8th Conference of the European Foundation for Plant Pathology & British Society of Plant Pathology Presidential Meeting, 2006 Copenhagen, Denmark

Publications arising from my PhD and Postdoc period, for recent papers see Tab "publications"

Papers <2002

• C. F. de Jong, F. L. W. Takken , X. Cai, P. J. G. M. de Wit and M. H. A.J. Joosten (2002)Attenuation of Cf -mediated defenseresponses atelevated temperatures correlates withadecrease in elicitor-binding sites.Molecular Plant-Microbe Interactions 15: 10. 1040-9.
• P. J. G. M. de Wit, B. F. Brandwagt, H. A. Van den Burg, X. Cai, R. A. L. Van der Hoorn, C.F. De Jong, J. van 't Klooster, M. J. D. de Kock, M. Kruijt, W. H. Lindhout, R. Luderer, F. L. W. Takken , N. Westerink, J.J. M. vervoort and M. H. A. J. Joosten (2002) The molecular basis of co-evolution between Cladosporium fulvum  and tomato. Antonie vanLeeuwenhoek 81: 409-412
• R. Luderer, F. L. W. Takken , P.J. G.m.Witand M. H. A. J. Joosten (2002) Cladosporiumfulvum overcomes Cf-2 -mediated resistance by producing truncated AVR2 elicitor proteins. Molecular Microbiology 45: 3. 875-84.
• W. I. L. Tameling, S.D. Elzinga,P.S. Darmin, J. H. Vossen, F. L. W. Takken , M.A.Haring and B. J. C. Cornelissen (2002) The tomato R gene products I-2 and Mi-1 are functional ATP binding proteins withATPase activity. Plant Cell 14: 11. 2929-39.
• X. Cai, F. L. W. Takken , M. H. A. J. Joosten and P.J. G. M. De Wit(2001) Specific recognition of Avr4 and Avr9 result in distinct patternsof hypersensitive cell death in tomato, but similar patterns ofdefence-related geneepxression. Molecular Plant Pathology 2: 2. 77-86
• B. F. Brandwagt, L. A.Mesbah, F. L. W. Takken , P. L. Laurent, T. J. A. Kneppers, J. Hille and H. J. J. Nijkamp (2000) A longevity assurance gene homolog of tomatomediates resistanceto Alternaria alternata f. sp. lycopersici toxins and fumonisin B1. Proc Natl Acad Sci USA 97:9.4961-6.
• F.L. W.Takken and M. H. A. J.Joosten (2000) Plant resistance genes: their structure, function and evolution. European Journal of Plant Pathology 106: 8. 699-713
• F. L. W. Takken, R. Luderer, S. H. E. J. Gabriëls, N.Westerink, R. Lu, P.J.G. M. de Wit and M.H.A.J.Joosten (2000) A functional cloning strategy, based on a binaryPVX-expression vector, toisolate HR-inducing cDNAsof plant pathogens. The Plant Journal 24: 2). 275-283
• L. A.Mesbah, T.J. A. Kneppers, F. L. W. Takken, P. Laurent,J.Hille and H. J. J. Nijkamp (1999) Geneticand physical analysis of aYAC contig spanning the fungal diseaseresistancelocus Asc of tomato ( Lycopersicon esculentum ). Molecular and General Genetics 261 (1): 50-57
• F.L. W. Takken , M. Thomas, M.H. A. J. Joosten, C. Golstein, N.Westerink, J. Hille, H. J. J. Nijkamp, P. J. G. M. DeWit and J. D. G. Jones (1999) A second gene at the tomato Cf-4 locus confers resistance to Cladosporium fulvum through recognition of anovel avirulence determinant. The Plant Journal 20: 3. 279-288
• F.L. W. Takken , D. Schipper, H. J. J. Nijkamp and J.Hille (1998)Identification and Ds-tagged isolation of a new gene at the Cf-4 locus of tomato involved in disease resistance to Cladosporium fulvum race 5. The Plant Journal 14: 401-411
  

• Takken, F.L.W., &Wit, P.J.G.M.de (10-01-2002). Elicitor from Cladosporium . no WO02/02787.
• Turk, S.C.H.J., Takken, F.L.W., Jong, C.F. de, Joosten, M.H.A.J.,& Wit, P.J.G.M. de (02-01-2003). Nucleotide sequences involved in plantdisease resistance.no01202420.4./ WO03000930  
• Joosten, M.H.A.J., Jong, C.F. de, Wit, P.J.G.M. de, Takken, F.L.W., & Turk, S.C.H.J. (17-03-2004). Nucleotide sequences involved in plantresistance. no EP1397515

Biography

Frank Takken (1969) is interested in how plants defend themselves against pathogens. Plants are able to sense the presence of invading microbes. Recognition subsequently results in activation of the plant innate immune system. In this process, resistance proteins play a key role as they mediate pathogen recognition and trigger the activation of downstream signalling cascades that halt the pathogen and thereby prevent disease.
Resistance genes come in different flavors, but the majority encodes intracellular tri-partite proteins that contain a central nucleotide binding domain. One of Frank's major research interests is the role the distinct domains in these proteins have for their function. Structure-function analysis in combination with 3D modelling of the different subdomain is used to predict mutations that will have specific effects on the activity of these proteins. These mutants are subsequently analyzed foraltered biochemical properties, differences in inter- and intramolecular interactions as well as for their effect on disease resistance.
To examine how resistance proteins activate downstream signalling it is essential to know their interacting partners. Using various screens, candidate interacting proteins have been identified and these proteins are analyzed for their involvement in plant defense.
Since April 2001 Frank is appointed as Assistant professor in the Molecular Plant Pathology group at the Swammerdam Institute for Life Sciences. Before he worked as a postdoc at the Phytopathology group at Wageningen University. During this time he worked on a self-written project at the biotech company Keygene NV in Wageningen. In that project he focused on the identification of genes that are transcriptionally regulated during the onset of plant defense signalling. He received his PhD in 1999 from the Free University of Amsterdam for his pioneering work on the isolation of resistance genes from tomato.

A resistant tomato plant carrying I-2 (left) and susceptible one (right side) infected with Fusarium oxysporum f.sp. l ycopersici.

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