Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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    Recognition of Verticillium effector Ave1 by tomato immune receptor Ve1 mediates Verticillium resistance in diverse plant species
    Song, Yin - \ 2017
    Wageningen University. Promotor(en): B.P.H.J. Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789463437950 - 231
    disease resistance - defence mechanisms - immunity - plant-microbe interactions - plant pathogens - verticillium dahliae - verticillium - tomatoes - solanum lycopersicum - receptors - genes - tobacco - nicotiana glutinosa - potatoes - solanum tuberosum - solanum torvum - humulus lupulus - cotton - gossypium hirsutum - transgenic plants - arabidopsis thaliana - ziekteresistentie - verdedigingsmechanismen - immuniteit - plant-microbe interacties - plantenziekteverwekkers - verticillium dahliae - verticillium - tomaten - solanum lycopersicum - receptoren - genen - tabak - nicotiana glutinosa - aardappelen - solanum tuberosum - solanum torvum - humulus lupulus - katoen - gossypium hirsutum - transgene planten - arabidopsis thaliana

    Plant-pathogenic microbes secrete effector molecules to establish disease on their hosts, whereas plants in turn employ immune receptors to try and intercept such effectors in order to prevent pathogen colonization. Based on structure and subcellular location, immune receptors fall into two major classes; cell surface-localized receptors that comprise receptor kinases (RKs) and receptor-like proteins (RLPs) that monitor the extracellular space, and cytoplasm-localized nucleotide-binding domain leucine-rich repeat receptors (NLRs) that survey the intracellular environment. Race-specific resistance to Verticillium wilt in tomato (Solanum lycopersicum) is governed by the tomato extracellular leucine-rich repeat (eLRR)-containing RLP-type cell surface receptor Ve1 upon recognition of the effector protein Ave1 that is secreted by race 1 strains of the soil-borne vascular wilt Verticillium dahliae. Homologues of V. dahliae Ave1 (VdAve1) are found in plants and in a number of plant pathogenic microbes, and some of these VdAve1 homologues are recognized by tomato Ve1. The research presented in this thesis aims to characterize the role of the tomato cell surface-localized immune receptor Ve1, and its homologues in other diverse plant species, in Verticillium wilt resistance.

    Evasion of chitin-triggered immunity by fungal plant pathogens
    Rövenich, Hanna J. - \ 2017
    Wageningen University. Promotor(en): B.P.H.J. Thomma; P.J.G.M. de Wit. - Wageningen : Wageningen University - ISBN 9789463436137 - 133
    plant-microbe interactions - immunity - receptors - verticillium dahliae - cladosporium - plant pathogens - chitin - arabidopsis thaliana - fungi - plant-microbe interacties - immuniteit - receptoren - verticillium dahliae - cladosporium - plantenziekteverwekkers - chitine - arabidopsis thaliana - schimmels

    Plants establish intricate relationships with microorganisms that range from mutualistic to pathogenic. In order to prevent colonization by potentially harmful microbes, plant hosts employ surface-localized receptor molecules that perceive ligands, which are either microbe-derived or result from microbe-mediated plant manipulation. This recognition ultimately leads to the activation of host immunity. In order to circumvent recognition or suppress immune responses, microbes secrete effector proteins that deregulate host physiological processes. While the number of identified putative effectors has rapidly increased in recent years, their functions and the mechanisms governing their recognition have largely remained unexplored. To enhance our understanding of the molecular interplay between host and microbe, the work presented here was designed to identify further components involved in the recognition of the two fungal pathogens Verticillium dahliae and Cladosporium fulvum, as well as to characterize the functions of effector proteins produced by these pathogens during tomato infection.

    Innate immunity of carp : fishing for receptors
    Fink, Inge - \ 2017
    Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789463430753 - 240
    carp - cyprinus - immunity - platelets - macrophage activation - receptors - polarization - immunostimulation - immunology - karper - cyprinus - immuniteit - bloedplaatjes - macrofaag activering - receptoren - polarisatie - immunostimulatie - immunologie

    Recent decades have seen a significant intensification of aquaculture leading to increased risk of infections with several pathogenic organisms. On economical and ethical grounds it is more appropriate to improve general welfare conditions and prevent infections rather than treating disease outbreaks once they have occurred. Immunostimulation through feed can provide more efficient and sustainable control of diseases in aquaculture through enhancing the immunocompetence of fish; however, the underlying mechanisms are poorly characterized. The overall aim of this thesis was to perform a molecular and functional characterization of how pathogen-associated molecular patterns (PAMPs), such as β-glucans, affect the innate immune response of carp and which receptors on carp leukocytes are likely candidates to play a role in sensing such PAMPs.

    In chapter 1 we provide a framework for this thesis by introducing different classes of PAMPs, including β-glucans. These molecules were the centrepiece of an intra-European training network called NEMO (Protective immune modulation in warm water fish by feeding glucans), which this PhD project was part of. The scientific aim of the NEMO network was to develop a sustainable and cost-effective use of β-glucans as immunostimulants for aquaculture, using common carp as the model fish species, since on a global scale common carp is the most cultured fish species for food consumption. Our aims within the NEMO project entailed both the characterization of carp leukocytes and the characterization of candidate pattern recognition receptors (PRRs) that could play a role in sensing PAMPs and initiating immune responses. Chapter 1 therefore introduces the thrombocytes and macrophages pertinent to this thesis, as well as important classes of PRRs.

    In our first experimental study, described in chapter 2, we investigated the relevance of thrombocytes for the immune system of carp. We found that thrombocytes from healthy carp express a large number of immune-relevant genes, among which several cytokines and Toll-like receptors (Tlrs). Furthermore, we dissected the role of thrombocytes during infections with two different, albeit related, protozoan parasites, Trypanoplasma borreli and Trypanosoma carassii, and found thrombocytes were massively depleted from blood and spleen of fish infected with T. borreli. The pathology of this infection is associated with elevated levels of tissue nitration, prompting us to investigate, ex vivo, the effect of nitric oxide on thrombocytes. Our studies revealed that nitric oxide can induce a clear and rapid apoptosis of thrombocytes from healthy carp, supporting a role for nitric oxide-mediated control of immune-relevant thrombocytes during infection with T. borreli. Thereby, this particular study provided an excellent example of interplay between pathogen and the innate immune system of carp.

    We reviewed in chapter 3 another cell type central to innate immunity: the macrophage. We focused on the heterogeneity of macrophage activation states as these cells, at least in humans and mice, have the ability to polarize in several directions during an immune response. Based on the signals that lead to activation and the effector functions and cytokine profile as a result thereof, macrophages can be broadly divided into two types: classically activated macrophages induced in a T helper 1 (TH1) cytokine environment, and alternatively activated macrophages, induced in a TH2 cytokine environment. Mirroring the TH1–TH2 dichotomy, classically activated macrophages have also been termed M1, whereas alternatively activated macrophages have been termed M2. Classically activated macrophages are typically induced by stimulation with microbial ligands such as LPS in combination with pro-inflammatory cytokines such as IFNγ, and can be viewed as an extension of innate activated macrophages which are induced by microbial ligands only, thus are independent of cytokines. Alternatively activated macrophages are generated in the presence of IL4 and/or IL13. In addition to M1 and M2, one can distinguish regulatory macrophages, which are associated with the presence of the cytokine IL10. In this chapter, we reviewed the evidence of existence of polarized macrophages in teleost fish, among other things based on observations of the fundamentally different immune responses elicited by the parasites T. borreli and T. carassii.

    We further investigated the polarization of carp macrophages in chapter 4, where we obtained gene signature profiles of carp macrophages via a transcriptome approach. Independently of cytokines, carp macrophages showed the ability to differentiate into cells with functional characteristics highly comparable to those of mammalian M1 and M2, consistent with a conserved ability of macrophages to polarize into distinct subsets. In addition to obtaining a global view of gene expression, our transcriptome approach identified gene signatures for M1 and M2 macrophages which appear conserved from fish to mammals. We selected a number of these interesting genes that were differentially regulated between M1 and M2 macrophages and discussed in detail five potential M1 markers; il1β, ptx3a, saa, nos2b, and il12a – as well as five potential M2 markers; cyr61, inhba, timp2, tgm2, and arg2. These transcriptome studies may pave the way for future studies of polarized macrophages during immune responses in fish. Furthermore, additional analyses of the datasets described in this chapter will undoubtedly lead to the characterization of more genes relevant to macrophage polarization and recognition of immunostimulants.

    As part of the characterization of candidate PRRs that could play a role in sensing PAMPs and initiating immune responses, we studied the scavenger receptor Cd36 (chapter 5), which in mammals is expressed by many different (immune) cell types and plays a role in highly diverse processes, both homeostatic and pathologic. Among other things, it is often found associated with sensing of β-glucans and also with M2 macrophage activation, sparking our interest in this molecule in fish. We studied Cd36 in common carp as well as in zebrafish, a closely related cyprinid fish species. Whereas a single cd36 gene is present in zebrafish, carp was shown to have two paralogs of cd36. Although all genes show conserved synteny compared to mammalian CD36, unexpectedly we could not detect gene expression of cyprinid cd36 in macrophages or any other immune cell type or immune organ. Yet, because gene expression of cd36 was down-regulated during Mycobacterium marinum infection of zebrafish, and knockdown of cd36 in zebrafish embryos led to higher bacterial burden upon such infection, our data imply a role for Cd36 in immune responses of fish. Future studies are needed to clarify the exact mechanisms involved.

    As characterization of candidate PRRs we also examined the Toll-like receptors Tlr1 and Tlr2 (chapter 6). We identified a full-length, expressed tlr1 gene, a tlr1 pseudogene, and a second tlr2 gene next to the tlr2 which had been described previously. Sequence, phylogenetic and synteny analyses supported the conserved nature of these genes, and three-dimensional modelling showed a good fit with the mammalian TLR1/TLR2 heterodimer including the potential to bind to the prototypical ligand Pam3CSK4. However, we were unable to demonstrate Tlr1/Tlr2-mediated ligand binding in transfected cell lines through NFκB activation, despite showing the expression and co-localization of Tlr1 and Tlr2. This prompted a discussion of methods available for studying ligand-binding properties of fish Tlrs.

    Finally, we discuss in chapter 7 the findings of this thesis in the context of the NEMO project. We present the concept of trained immunity, which could provide the conceptual framework within which the immune-stimulating ability of compounds such as β-glucans could be explained. We discuss recent advances in the field of TLR research as well as that of macrophage polarization, and highlight immunometabolism as a new area of interest which may help to illuminate the molecular events occurring in immune cells during health and disease. In conclusion, we found that carp leukocytes, along with their pattern recognition receptors, are central players of the innate immune system of carp. Our findings contribute to the understanding of mechanisms of immunostimulation, and expect this will enable the valorisation and use of immunostimulants for sustainable aquaculture and improvement of fish health.

    High fat challenges with different fatty acids affect distinct atherogenic gene expression pathways in immune cells from lean and obese subjects
    Esser, D. ; Dijk, S.J. van; Oosterink, E. ; Lopez, S. ; Muller, M.R. ; Afman, L.A. - \ 2015
    Molecular Nutrition & Food Research 59 (2015)8. - ISSN 1613-4125 - p. 1563 - 1572.
    triglyceride-rich lipoproteins - blood mononuclear-cells - men - atherosclerosis - inflammation - activation - receptors - adherence - profiles - alpha
    Scope - Early perturbations in vascular health can be detected by imposing subjects to a high fat (HF) challenge and measure response capacity. Subtle responses can be determined by assessment of whole-genome transcriptional changes. We aimed to magnify differences in health by comparing gene-expression changes in peripheral blood mononuclear cells toward a high MUFA or saturated fatty acids (SFA) challenge between subjects with different cardiovascular disease risk profiles and to identify fatty acid specific gene-expression pathways. Methods and results -In a cross-over study, 17 lean and 15 obese men (50–70 years) received two 95 g fat shakes, high in SFAs or MUFAs. Peripheral blood mononuclear cell gene-expression profiles were assessed fasted and 4-h postprandially. Comparisons were made between groups and shakes. During fasting, 294 genes were significantly differently expressed between lean and obese. The challenge increased differences to 607 genes after SFA and 2516 genes after MUFA. In both groups, SFA decreased expression of cholesterol biosynthesis and uptake genes and increased cholesterol efflux genes. MUFA increased inflammatory genes and PPAR-a targets involved in ß-oxidation. Conclusion - Based upon gene-expression changes, we conclude that an HF challenge magnifies differences in health, especially after MUFA. Our findings also demonstrate how SFAs and MUFAs exert distinct effects on lipid handling pathways in immune cells.
    Understanding the role of L-type lectin receptor kinases in Phytophthora resistance
    Wang, Y. - \ 2014
    Wageningen University. Promotor(en): Francine Govers, co-promotor(en): W. Shan; Klaas Bouwmeester. - Wageningen : Wageningen University - ISBN 9789462571327 - 214
    phytophthora - phytophthora capsici - oömycota - plantenziekteverwekkende schimmels - plant-microbe interacties - arabidopsis - transgene planten - genexpressie - receptoren - kinasen - genen - ziekteresistentie - immuniteit - phytophthora - phytophthora capsici - oomycota - plant pathogenic fungi - plant-microbe interactions - arabidopsis - transgenic plants - gene expression - receptors - kinases - genes - disease resistance - immunity

    Abstract

    Phytophthora pathogens are notorious for causing severe damage to many agriculturally and ornamentally important plants. Effective plant resistance depends largely on the capacity to perceive pathogens and to activate rapid defence. Cytoplasmic resistance (R) proteins are well-known for activation of plant immunity upon recognition of matching effectors secreted by Phytophthora. However, Phytophthora pathogens are notoriously difficult to control due to their rapid adaptation to evade R protein-mediated recognition. Hence, exploring novel resistance components is instrumental for developing durable resistance. Receptor-like kinases (RLKs) function as important sentinels in sensing exogenous and endogenous stimuli to initiate plant defence. One RLK that was previously identified as a novel Phytophthora resistance component is the Arabidopsis L-type lectin receptor kinase LecRK-I.9. This RLK belongs to a multigene family consisting of 45 members in Arabidopsis but whether or not the other members function in Phytophthora resistance was thus far unknown. The research described in this thesis was aimed at unravelling the role of LecRKs in plant immunity, in particular to Phytophthora pathogens.

    Chapter I describes various Phytophthora diseases and the current understanding of the mechanisms underlying plant innate immunity with emphasis on disease resistance to Phytophthora pathogens.

    In Chapter II, we describe the development of a new Arabidopsis-Phytophthora pathosystem. We demonstrated that Phytophthora capsici is capable to infect Arabidopsis. Inoculation assays and cytological analysis revealed variations among Arabidopsis accessions in response to different P. capsici isolates. Moreover, infection assays on Arabidopsis mutants with specific defects in defence showed that salicylic acid signaling, camalexin and indole glucosinolates biosynthesis pathways are required for P. capsici resistance (Chapter IIa). The importance of these pathways in Arabidopsis resistance was supported by the finding that the corresponding marker genes are induced upon infection by P. capsici (Chapter IIb). This model pathosystem can be used as an additional tool to pinpoint essential components of Phytophthora resistance.

    We then exploited Arabidopsis-Phytophthora pathosystems to uncover the role of LecRKs in Phytophthora resistance. In Chapter III we describe a systematic phenotypic characterization of a large set of Arabidopsis LecRK T-DNA insertion lines. The T-DNA insertion lines were assembled and assayed for their response towards different Phytophthora pathogens. This revealed that next to LecRK-I.9, several other LecRKs function in Phytophthora resistance. We have also analysed whether the LecRKs are involved in response to other biotic and abiotic stimuli. Several T-DNA insertion lines showed altered responses to bacterial or fungal pathogens, but none of the lines showed visible developmental changes under normal conditions or upon abiotic stress treatment. Combining these phenotypic data with LecRK expression profiles obtained from publicly available datasets revealed that LecRKs that are hardly induced or even suppressed upon infection, might still have a function in pathogen resistance. Computed co-expression analysis revealed that LecRKs with similar function display diverse expression patterns.

    Arabidopsis LecRK clade IX comprises two members. T-DNA insertion mutants of both LecRK-IX.1 and LecRK-IX.2 showed gain of susceptibility to non-adapted Phytophthora isolates and therefore the role of these two LecRKs in Phytophthora resistance was further investigated. In Chapter IV we describe that overexpression of either LecRK-IX.1 or LecRK-IX.2 in Arabidopsis resulted in increased resistance to Phytophthora, but also induced plant cell death. A mutation in the kinase domain abolished the ability of LecRK-IX.1 and LecRK-IX.2 to induce Phytophthora resistance as did deletion of the lectin domain. Cell death induction however, only required the kinase, not the lectin domain. Since transient expression of both LecRKs in Nicotiana benthamiana also resulted in increased Phytophthora resistance and induction of cell death, we used N. benthamiana to explore downstream components required for LecRK-IX.1- and LecRK-IX.2-mediated Phytophthora resistance and cell death. Virus-induced gene silencing of candidate signaling genes revealed that NbSIPK1/NPT4 is essential for LecRK-IX.1-mediated cell death but not for Phytophthora resistance. Collectively, these results illustrate that the Phytophthora resistance mediated by LecRK-IX.1 and LecRK-IX.2 is independent of the cell death phenotype. By co-immunoprecipitation we identified putative interacting proteins, one of which was an ATP-binding cassette (ABC) transporter. A homolog in Arabidopsis, the ABC transporter ABCG40, was found to interact in planta with both LecRK-IX.1 and LecRK-IX.2. Similar to the LecRK mutants, Arabidopsis ABCG40 mutants showed compromised Phytophthora resistance, indicating that ABCG40 has a function in Phytophthora resistance.

    In Chapter V, we describe the generation of stable transgenic N. benthamiana plants expressing Arabidopsis LecRK-I.9 or LecRK-IX.1. Multiple transgenic lines were obtained varying in transgene copy number and transgene expression level. Ectopic expression of LecRK-I.9 resulted in reduced plant sizes and aberrant leaf morphology. In addition, expression of LecRK-IX.1 induced plant cell death. Transgenic N. benthamiana lines expressing either LecRK-I.9 or LecRK-IX.1 showed increased resistance towards P. capsici or Phytophthora infestans. This demonstrated that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their role in Phytophthora resistance upon interfamily transfer.

    Based on the results obtained on Arabidopsis LecRKs, we speculated that LecRKs in other plant species could play a similar role in Phytophthora resistance. In Chapter VI, we focus on LecRKs in two Solanaceous plants, i.e. N. benthamiana and tomato. By exploring genome databases, we identified 38 and 22 LecRKs in N. benthamiana and tomato, respectively. Phylogenetic analysis revealed that both N. benthamiana and tomato lack LecRKs homologous to Arabidopsis LecRKs of clades I, II, III and V, but contain a Solanaceous-specific clade of LecRKs. Functional analysis of various Solanaceous LecRKs using virus-induced gene silencing followed by infection assays revealed that homologs of Arabidopsis LecRK-IX.1 and LecRK-IX.2 in N. benthamiana and tomato are implicated in Phytophthora resistance. These results indicate that the role of clade IX LecRKs in Phytophthora resistance is conserved across plant species.

    In Chapter VII, the experimental data presented in this thesis are summarized and discussed in a broader context. We present an overview of the current understanding of LecRKs in plant immunity and discuss how LecRKs can be exploited to improve plant resistance.

    Role of Frizzled6 in the molecular mechanism of beta-carotene action in the lung
    Piga, R. ; Dartel, D.A.M. van; Bunschoten, A. ; Stelt, I. van der; Keijer, J. - \ 2014
    Toxicology 320 (2014). - ISSN 0300-483X - p. 67 - 73.
    epidemiologic evidence - rna interference - cell-cycle - cancer - receptors - mice - identification - prevention - aneuploidy - expression
    ß-Carotene (BC) is omnipresent in our diet, both as natural food component as well as an additive. BC and its metabolites have important biological functions. For this reason, BC is generally considered to be a health promoting compound. Two human trials, however, have described adverse effects in lung tissue, increasing the risk of lung cancer. We previously applied transcriptomic analyses in a unique animal model, beta-carotene 15,15'-monooxygenase 1 knockout (Bcmo1-/-) mice that are, like humans, able to accumulate intact BC. In our search to unravel the molecular action of BC in the lung, we previously identified two genes particularly strongly down-regulated by BC in lung tissue of the male Bcmo1-/- mice: frizzled homologue 6 (Fzd6) and collagen triple helix repeat containing 1 (Cthrc1). In the present study, our aim was to further elucidate the role of FZD6 in lung epithelial cells and to provide a mechanistic explanation for BC increased lung cancer risk in humans. We performed whole genome microarray analysis on silenced FZD6 in non-tumor human type II bronchial epithelial BEAS-2B cells using RNAi. To directly link FZD6 to BC-effects on the lung, we compared the FZD6-silenced BEAS-2B gene expression profile to the BC-dependent gene expression profile of Bcmo1-/- mouse lungs. A number of relevant genes were regulated in the same direction in FZD6- BEAS-2B and in BC-exposed lungs of Bcmo1-/- mice and revealed enrichment of the Gene Ontology terms “oncogenes”, “cell proliferation” and “cell cycle”, which suggests a mediating role of FZD6 in BC-induced uncontrolled proliferation of lung cells.
    Fine mapping of the gene Rvi18 (V25) for broad-spectrum resistance to apple scab, and development of a linked SSR marker suitable for marker-assisted breeding
    Soriano, J.M. ; Madduri, M. ; Schaart, J. ; Burgh, A.M. van der; Kaauwen, M.P.W. van; Tomic, L. ; Groenwold, R. ; Velasco, R. ; Weg, W.E. van de; Schouten, H.J. - \ 2014
    Molecular Breeding 34 (2014)4. - ISSN 1380-3743 - p. 2021 - 2032.
    malus-x-domestica - venturia-inaequalis - microsatellite markers - cisgenic plants - genome - borkh. - receptors - lectins - race
    Apple scab, caused by the fungal pathogen Venturia inaequalis, is one of the most devastating diseases for the apple growing industry in temperate zones with humid springs and summers. Breeding programs around the world have identified several sources of resistance, of which the Rvi6 (Vf) gene from Malus floribunda 821 has been the most widely used. The appearance of Rvi6-virulent strains of V. inaequalis in several European countries have underlined the necessity of pyramiding different effective resistance genes for durably resistant cultivars. Here we report the mapping of the new apple scab resistance gene Rvi18 (V25) from the selection 1980-015-025 of the apple breeding program at Wageningen University and Research Centre, The Netherlands. This gene was fine mapped on the proximal part of LG11 to a region of 34 Kb in the apple genome sequence of ‘Golden Delicious’, using 894 progeny plants, and SSR, DArT, AFLP, and SNP markers. One gene on the ‘Golden Delicious’ reference genome was identified as the potential susceptibility allele of the resistance gene. Moreover, an SSR marker has been developed of which one of its amplicons sizes is highly specific for Rvi18, thus facilitating the directed pyramiding of resistance genes through marker assisted breeding.
    Human protein status modulates brain reward responses to food cues1–3
    Griffioen-Roose, S. ; Smeets, P.A.M. ; Heuvel, E.M. van den; Boesveldt, S. ; Finlayson, G. ; Graaf, C. de - \ 2014
    American Journal of Clinical Nutrition 100 (2014)1. - ISSN 0002-9165 - p. 113 - 122.
    leverage hypothesis - energy-intake - taste - breakfast - appetite - satiety - carbohydrate - mechanisms - receptors - choice
    Background: Protein is indispensable in the human diet, and its intake appears tightly regulated. The role of sensory attributes of foods in protein intake regulation is far from clear. Objective: We investigated the effect of human protein status on neural responses to different food cues with the use of functional magnetic resonance imaging (fMRI). The food cues varied by taste category (sweet compared with savory) and protein content (low compared with high). In addition, food preferences and intakes were measured. Design: We used a randomized crossover design whereby 23 healthy women [mean SD age: 22 +/- 2 y; mean +/- SD body mass index (in kg/m(2)): 22.5 +/- 1.8] followed two 16-d fully controlled dietary interventions involving consumption of either a low-protein diet (0.6 g protein center dot kg body weight(-1) center dot d(-1), similar to 7% of energy derived from protein, approximately half the normal protein intake) or a high-protein diet (2.2 g protein center dot kg body weight(-1) center dot d(-1), similar to 25% of energy, approximately twice the normal intake). On the last day of the interventions, blood oxygen level-dependent (BOLD) responses to odor and visual food cues were measured by using fMRI. The 2 interventions were followed by a 1 -d ad libitum phase, during which a large array of food items was available and preference and intake were measured. Results: When exposed to food cues (relative to the control condition), the BOLD response was higher in reward-related areas (orbitofrontal cortex, striatum) in a low-protein state than in a high-protein state. Specifically, BOLD was higher in the inferior orbitofrontal cortex in response to savory food cues. In contrast, the protein content of the food cues did not modulate the BOLD response. A low protein state also increased preferences for savory food cues and increased protein intake in the ad libitum phase as compared with a high-protein state. Conclusions: Protein status modulates brain responses in reward regions to savory food cues. These novel findings suggest that dietary protein status affects taste category preferences, which could play an important role in the regulation of protein intake in humans. This trial was registered at www.trialregister.nl/trialreg/admin/rctview.asp?TC=3288 as NTR3288.
    Venom allergen-like proteins in secretions of plant-parasitic nematodes activate and suppress extracellular plant immune receptors
    Lozano Torres, J.L. - \ 2014
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - Wageningen University - ISBN 9789461739193 - 201
    plantenparasitaire nematoden - nematoda - receptoren - vergiffen - eiwitten - secretie - immuniteit - modulatie - plant parasitic nematodes - nematoda - receptors - venoms - proteins - secretion - immunity - modulation

    Parasitic worms threaten human, animal and plant health by infecting people, livestock and crops worldwide. Animals and plants share an anciently evolved innate immune system. Parasites modulate this immune system by secreting proteins to maintain their parasitic lifestyle. This thesis describes how venom-allergen-like proteins (VAPs) that both animal- and plant-parasitic nematodes release into their hosts, modulate host innate immunity. On the one hand we found that one particular secreted VAP from the potato cyst nematode can activate host defenses in tomato plants, opening an opportunity for plant breeders to generate novel nematode-resistant cultivars. We showed that plants make more efficiently use of their limited repertoire of immune receptors by guarding common virulence targets of multiple unrelated plant pathogens. While on the other hand, we describe how VAPs may be used by parasites to suppress the host defense responses mediated by extracellular immune receptors. In short, this fundamental study contributes to our understanding of the molecular basis of persistent infections by parasitic nematodes in plants and in animals.

    Functional characterization of the PPAR targets ANGPTL4 and HILPDA in lipid metabolism
    Mattijssen, F.B.J. - \ 2014
    Wageningen University. Promotor(en): Sander Kersten. - Wageningen : Wageningen University - ISBN 9789461739087 - 193
    lipidenmetabolisme - receptoren - genotype-voeding interactie - lipid metabolism - receptors - genotype nutrition interaction

    The peroxisomal proliferator activator receptors (PPARs) are ligand-activated transcription factors that play important roles in the regulation of lipid metabolism. Three PPAR isoforms have been identified: PPARα, PPARβ/δ, and PPARγ. Each isoform has specific functions determined by their relative abundance in a cell as well as ligand specificity.

    A highly sensitive PPAR target gene is represented by Angiopoietin-like 4 (ANGPTL4), which was discovered by two independent groups in 2000. ANGPTL4 is produced in a number of organs including liver and adipose tissue where its expression is governed by PPARα and PPARγ, respectively. Upon secretion, ANGPTL4 is cleaved into n- and c-terminal fragments that have divergent functions. nANGPTL4 is known to function as an inhibitor of lipoprotein lipase and hepatic lipase, whereas cANGPTL4 is involved in a number of processes including tumorigenesis and wound healing and is known to interact with integrins β1 and β5.

    In this thesis we set out to expand our knowledge on the molecular function of ANGPTL4 in the regulation of lipid metabolism. We used a variety of animal models, cell culture, biochemical assays, and other functional measurements to zoom in on previously unexplored aspects of ANGPTL4.

    Feeding mice deficient in ANGPLT4 a diet rich in long-chain saturated fatty acids elicited a complex phenotype and Angptl4-/- mice ultimately died from fibrinopurulent peritonitis. In contrast, the prevalence of the lethal phenotype was absent when the fat component of the high-fat diet was changed to medium-chain fatty acids, suggesting a role for increased chyle flow. Indeed, Angptl4-/- mice had dramatically enlarged mesenteric lymph nodes which contained numerous lipid laden macrophages. In vitro experiments showed that PPARβ/δ mediated induction of ANGPTL4 inhibits macrophage LPL. In the absence of ANGPTL4 there is increased lipid uptake in mesenteric lymph node macrophages, leading to ER stress and subsequent inflammatory response.

    Additionally, Angptl4-/- mice gain more weight when fed a high-fat diet containing mainly unsaturated fatty acids. The increased body weight and adiposity was unrelated to food intake, activity, or energy expenditure. Remarkably, we observed increased lipid digestion in Angptl4-/- mice, which coincided with increased luminal lipase activity in the intestines of Angptl4-/- mice. Using biochemical assays we reveal that ANGPTL4 inhibits pancreatic lipase.

    In the second part of this thesis we identified a novel PPAR target gene, hypoxia inducible lipid droplet associated (HILPDA). We observed HILPDA expression to be increased in liver slices exposed to a synthetic PPARα ligand. Additionally, oral dosing of similar ligand induced a marked increase in Hilpda expression in wild-type mice but not in Pparα-/- mice. PPAR mediated induction of Hilpda expression was found to be mediated by a conserved and functional PPRE located 1200 base pair or the transcription start site of HILPDA. Functional characterization of HILPDA in liver was performed via adeno-associated virus mediated overexpression. Interestingly, increased hepatic expression of HILPDA was associated with the development of a fatty liver, which could be attributes to a decrease in hepatic VLDL production.

    HILPDA was also found to be highly expressed in both human and mouse adipose tissue, where its expression is under the control of PPARγ and β-adrenergic receptor. Moreover, adipose tissue HILPDA expression was increased with fasting and decreased with high-fat feeding. Despite the regulation of adipose tissue HILPDA by PPARγ, we observed no effect of HILPDA on adipogenesis. Furthermore, adipose tissue specific Hilpda knock-out mice showed no major metabolic perturbations upon fasting. However, overexpression of HILPDA in adipocytes significantly reduced the release of NEFA upon β-adrenergic receptor activation. Induction of HILPDA by β-adrenergic receptor stimulation may be part of feedback mechanism to regulate adipocyte lipolysis.

    In conclusion, in thesis we have extended the current knowledge on the function of ANGPTL4. We show that ANGPTL4 serves as an important regulator in the process of lipid digestion and also in the protection of macrophages that reside in mesenteric lymph nodes that are exposed to high concentrations of lipid. HILPDA is a novel PPAR target that is involved in hepatic VLDL secretion and adipocyte lipolysis. Future research will focus on elucidating the mechanistic aspects of the regulation and function of HILPDA.

    Intrinsic bitterness of flavonoids and isoflavonoids and masking of their taste activity
    Roland, W.S.U. - \ 2014
    Wageningen University. Promotor(en): Harry Gruppen; Gerrit Smit, co-promotor(en): Jean-Paul Vincken. - Wageningen : Wageningen University - ISBN 9789461738530 - 188
    flavonoïden - isoflavonoïden - bitterheid - receptoren - chemische structuur - flavonoids - isoflavonoids - bitterness - receptors - chemical structure

    Many flavonoids and isoflavonoids have been associated with beneficial health effects. Therefore, consumption of (iso)flavonoid-rich food products, and enrichment of foods with (iso)flavonoids is becoming increasingly popular. However, several (iso)flavonoids have been reported as bitter. Consequently, their incorporation in (or fortification of) foods can introduce (or enhance) bitterness. Hence, debittering strategies are demanded.

    Some (iso)flavonoids have unknown taste properties, as they have never been incorporated in high levels in food products. For other (iso)flavonoids, contradictory findings on bitterness have been made in sensory tests. Therefore, objective tests are necessary to identify which (iso)flavonoids contribute to bitterness of a food product. An objective tool to study bitterness is a cell-based bitter taste receptor assay. Twenty-five different bitter taste receptors (hTAS2Rs) occur on the human tongue, each of which has been introduced in a separate human embryonic kidney (HEK)293 cell line. With these, the “intrinsic bitterness” of a compound can be investigatedin vitro. Intrinsic bitterness is the capacity of a compound to activate bitter taste receptors, uncoupled from cross-modal interactions and interactions with salivary proteins and oral mucosa. The aim of this research was to study the intrinsic bitterness of a large set of (iso)flavonoids and to investigate structural requirements for (iso)flavonoids to activate the bitter receptors identified. A subsequent aim was the investigation of different debittering strategies by the use of the bitter receptor assay.

    Chapter 1provides an overview of flavonoids and isoflavonoids with respect to their structural classification, sensorial properties and occurrence as dietary compounds. Taste perception and the mode of action of bitter taste receptors are introduced. The measurement of bitter receptor activation in vitro is explained, as well as strategies to reduce bitter receptor activation, and bitter taste in general. A state-of-the-art overview of all 25 bitter taste receptors is given with respect to known agonists and antagonists.

    The aim of Chapter 2was to identify the bitter receptor(s) that recognize the bitter taste of the soy isoflavone genistein. Screening of all 25 human bitter receptors revealed genistein as agonist of hTAS2R14 and hTAS2R39. Genistein displayed threshold values of 4 and 8 µM on hTAS2R14 and hTAS2R39, and EC50 values of 29 and 49 µM, respectively. Besides, the behavior of structurally similar isoflavonoids was investigated. Although the two receptors are not closely related, the results for hTAS2R14 and hTAS2R39 were similar towards most isoflavonoid aglycones. Glucosylation of isoflavones seemed to inhibit activation of hTAS2R14, whereas four of five glucosylated isoflavones were agonists of hTAS2R39, namely glycitin, genistin, acetyl genistin, and malonyl genistin. A total of three hydroxyl substitutions of the A- and B-rings of the isoflavonoids seemed to be more favorable for receptor activation than less hydroxyl groups. The concentration of the trihydroxylated genistein in several soy foods exceeds the bitter receptor threshold values determined, whereas those of other soy isoflavones are around or below their respective threshold values. Despite its low concentration, genistein might be one of the main contributors to the bitterness of soy products. Furthermore, the bioactive isoflavonoids equol and coumestrol activated both receptors, indicating that their sensory impact should be considered when used as food ingredients.

    In Chapter 3, the intrinstic bitterness of (iso)flavonoids, which can hamper their use as food bioactives, was investigated further.The effect of a large set of structurally similar (iso)flavonoids on the activation of bitter receptors hTAS2R14 and hTAS2R39 was tested, and their structural requirements to activate these receptors were predicted. In total, 68 compounds activated hTAS2R14 and 70 compounds activated hTAS2R39, amongst which 58 ligands were overlapping. Their activation threshold values varied over a range of three log units between 0.12 and 500 μM. Ligand-based 2D-fingerprint and 3D-pharmacophore models were created to detect structure activity relationships. The 2D-models demonstrated excellent predictive power in identifying bitter (iso)flavonoids and discrimination from inactive ones. The structural characteristics for an (iso)flavonoid to activate hTAS2R14 and hTAS2R39 were determined by 3D-pharmacophore models to be composed of two (for hTAS2R14) or three (for hTAS2R39) hydrogen bond donor sites, one hydrogen bond acceptor site, and two aromatic ring structures, of which one had to be hydrophobic. An additional hydrogen bond donor feature for hTAS2R39 ligands indicated the possible presence of another complementary acceptor site in the binding pocket, compared to hTAS2R14. Hydrophobic interaction of the aromatic feature with the binding site might be of higher importance in hTAS2R14 than in hTAS2R39. Together, this might explain why OH-rich compounds showed different behavior towards the two bitter receptors. The combination of in vitro data and different in silico methods created a good insight in activation of hTAS2R14 and hTAS2R39 by (iso)flavonoids and provided a powerful tool in prediction of their potential bitterness. By understanding the “bitter motif”, introduction of bitter taste in functional foods enriched in (iso)flavonoid bioactives might be avoided.

    Bitter receptor hTAS2R39 is activated by many different classes of bitter compounds, amongst which (iso)flavonoids. Nevertheless, several flavanones are known to mask bitter taste sensorially, andnot all flavanones reported in Chapter 3 activated hTAS2R39. For that reason, in Chapter 4, fourteen flavanones were investigated for their potential to reduce activation of hTAS2R39 by epicatechin gallate (ECG), one of the main bitter compounds present in green tea.Three compounds showed inhibitory behavior towards the activation of hTAS2R39 by ECG: 4’-fluoro-6-methoxyflavanone, 6,3’-dimethoxyflavanone, and 6-methoxyflavanone (in order of decreasing potency). The 6-methoxyflavanones also inhibited activation of hTAS2R14 (another bitter receptor activated by ECG), though to a lesser extent. Dose-response curves of ECG at various concentrations of the most potent antagonist 4’-fluoro-6-methoxyflavanone and wash-out experiments indicated reversible insurmountable antagonism. The same effect was observed for the structurally different agonist denatonium benzoate, suggesting a non-competitive orthosteric mechanism. The bitter receptor blockers identified might not be applicable to food products. Nevertheless, they create insight into structural requirements, which might lead to other, more suitable, blockers.

    Chapter 5investigates another strategy to reduce bitterness, namely complexation of bitter flavonoids with food proteins. The binding characteristics of the bitter tea compound epigallocatechin gallate (EGCG) to purified food proteins, and their equivalent food-grade preparations, were related to their effects on reducing bitter receptor activation by EGCG in vitro and their bitter-masking potential in vivo. β-Casein, in particular, and several gelatins, are known as strong binders of EGCG, contrary to β-lactoglobulin. Also in the bitter receptor assay, β-casein showed the strongest effect, with a maximum reduction of hTAS2R39 activation of about 93%. A similar potency was observed for Na-caseinate, which was applied as food-grade alternative for β-casein. β-Lactoglobulin had little effect on bitter receptor activation, as expected based on its low binding affinity for EGCG. The bitter-masking potential of Na-caseinate was confirmed in vivo using a trained sensory panel. β-Lactoglobulin also slightly reduced EGCG bitter perception, which could not be directly related to its binding capacity. The bitter receptor assay appeared to be a valid tool to evaluate in vitro the efficacy of food proteins as complexing agents for bitter-masking.

    Chapter 6discusses the findings presented in this thesis, addresses prospects and limitations of the bitter receptor cell assay, presents additional results on testing (iso)flavonoids for possible antagonistic properties, and compares taste evaluation by sensory tests, receptor assays and modeling. Furthermore, it evaluates strategies for bitter taste reduction, and applies the findings to soy products and tea.

    The systematic investigation of (iso)flavonoid aglycones showed that the substitution pattern of (iso)flavonoids is of higher importance for bitter receptor activation than the backbone structure. In case of bitter receptor antagonists, the substitution pattern as well as backbone structure revealed to be crucial for functionality. The bitter receptor assay was shown to be an appropriate tool not only for identification of bitter receptor agonists and antagonists, but also for identification of reduced receptor activation by complexing agents. Based on the findings of this thesis, it was concluded that complexation with food proteins is the most promising strategy to reduce bitter taste of flavonoids in tea. On the other hand, for soybean isoflavones, debittering by use of bitter receptor blockers seemed to be a promising debittering strategy. Alternatively to the use of receptor blockers, processing conditions (leading to low isoflavone aglycone formation) or raw material choice (i.e. cultivars low in genistein forms) were recommended. In conclusion, the choice of debittering strategies depends on the molecular structure of the bitter food compounds, as exemplified for soybean products and tea. Therefore, each food product seems to require its own tailor-made debittering solution.

    Towards generating broad-spectrum resistance to pathogens in plants: studies on a down-stream signalling NB-LRR of tomato
    Sueldo, D.J. - \ 2014
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten; Wladimir Tameling. - Wageningen : Wageningen University - ISBN 9789461738974 - 209
    solanum lycopersicum - tomaten - ziekteresistentie - verdedigingsmechanismen - receptoren - pathogenesis-gerelateerde eiwitten - bindende eiwitten - virulentie - mutanten - genetische kartering - solanum lycopersicum - tomatoes - disease resistance - defence mechanisms - receptors - pathogenesis-related proteins - binding proteins - virulence - mutants - genetic mapping
    Biogenesis and signalling requirements of plant receptor-like proteins mediating resistance to fungal pathogens
    Liebrand, T.W.H. - \ 2014
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten. - Wageningen : Wageningen University - ISBN 9789461738622 - 192
    solanum lycopersicum - plantenziekteverwekkende schimmels - passalora fulva - verticillium dahliae - ziekteresistentie - verdedigingsmechanismen - receptoren - pathogenesis-gerelateerde eiwitten - genetische analyse - genexpressie - solanum lycopersicum - plant pathogenic fungi - passalora fulva - verticillium dahliae - disease resistance - defence mechanisms - receptors - pathogenesis-related proteins - genetic analysis - gene expression - cum laude
    cum laude graduation
    Bioactivity screening and mass spectrometric confirmation for the detection of PPAR-delta agonists that increase type 1 muscle fibres
    Bovee, T.F.H. ; Blokland, M.H. ; Kersten, A.H. ; Hamers, A.R.M. ; Heskamp, H.H. ; Essers, M.L. ; Nielen, M.W.F. ; Ginkel, L.A. van - \ 2014
    Analytical and Bioanalytical Chemistry 406 (2014). - ISSN 1618-2642 - p. 705 - 713.
    human skeletal-muscle - gamma - macrophages - expression - receptors - cells - acid - gene - fat
    Sensitive and robust bioassays able to detect nuclear receptor activation are very useful for veterinary and doping control, pharmaceutical industry and environmental scientists. Here, we used bioassays based on human leukemic monocyte lymphoma U937 and human liver hepatocellular carcinoma HepG2 cell lines to detect the ligand-induced activation of the peroxisome proliferator-activated receptor delta (PPARd). Exposure of U937 cells to the PPARd agonist GW501516 resulted in a marked increase in mRNA expression of the PPARd target gene Angptl4 which was quantified by qRTPCR analysis. Exposure ofHepG2 cells transiently transfected with a PPARd expression plasmid and a PPAR-response element-driven luciferase reporter plasmid to PPARd agonists GW501516, GW610742 and L-165041 resulted in clear dose–response curves. Although the qRT-PCR resulted in higher fold inductions, the luciferase assay with transfected HepG2 cells is cheaper and quicker and about ten times more sensitive to GW501516 compared to analysis of Angptl4 mRNA expression in U937 cells by qRT-PCR. The HepG2- based luciferase assay was therefore used to screen GW501516-spiked supplements and feed and water samples. After liquid extraction and clean-up by solid phase extraction using a weak anion exchange column, extracts were screened in the HepG2 bioassay followed by confirmation with a newly developed UPLC-MS/MS method, using two transitions for each compound, i.e., for GW501516, 454.07>188.15 (collision energy (CE) 46 V) and 454.07>257.08 (CE 30 V); for GW610742, 472.07>206.2 (CE 48 V) and 472.07>275.08 (CE 30 V); and for L-165041, 401.2>193.15 (CE 26 V) and 401.2>343.2 (CE 20 V).
    Effects of blood-feeding on olfactory sensitivity of the malaria mosquito Anopheles gambiae: application of mixed linear models to account for repeated measurements
    Qiu, Y.T. ; Gort, G. ; Torricelli, A. ; Takken, W. ; Loon, J.J.A. van - \ 2013
    Journal of Insect Physiology 59 (2013). - ISSN 0022-1910 - p. 1111 - 1118.
    l-lactic acid - host-seeking behavior - human-sweat - diptera-culicidae - aedes-aegypti - sensu-stricto - electrophysiological responses - limburger cheese - odor - receptors
    Olfaction plays an important role in the host-seeking behavior of the malaria mosquito Anopheles gambiae. After a complete blood meal, female mosquitoes will not engage in host-seeking behavior until oviposition has occurred. We investigated if peripheral olfactory sensitivity changed after a blood meal by recording electroantennograms (EAGs) of female mosquitoes at three time points (2h, 48 h and 72 h) to 15 volatile kairomones of either human origin or documented to emanate from oviposition sites. The EAG-sensitivity was compared with that of females of similar age post eclosion. As is common practice in electrophysiological studies, the EAG recordings were obtained by repeated stimulation of the same antennal preparations. We introduce mixed linear modeling as an improved statistical analysis for electrophysiological data. Two hours after blood ingestion, olfactory sensitivity as quantified through EAG-recording increased significantly and selectively, i.e. for seven compounds, compared to unfed females of the same age. Such short-term electrophysiological sensitization in the olfactory system as a result of feeding has not been documented before for insects. Sensitization to six compounds persisted until 48 h or 72 h post-blood meal at one or more concentrations. Desensitization was observed at 48 and 72 h pbm in response to two and three kairomones, respectively. For several compounds, sensitization at the EAG-level corresponded with sensitization found previously in single sensillum studies on olfactory neurons in antennal sensilla trichodea of An. gambiae females. These effects are likely to reflect sensitization to oviposition cues, as eggs have matured 48-72 h pbm. Knowledge of changes in olfactory sensitivity to kairomones can be applied to increase trap catches of malaria mosquitoes that have taken a blood meal and need to locate oviposition sites.
    Stimulation of the innate immune system of carp: role of Toll-like receptors
    Pietretti, D. - \ 2013
    Wageningen University. Promotor(en): Geert Wiegertjes; Huub Savelkoul, co-promotor(en): Maria Forlenza. - Wageningen : Wageningen University - ISBN 9789461737878 - 213
    karper - cyprinus - immuunsysteem - verdedigingsmechanismen - immuniteitsreactie - immunostimulatie - bèta-glucaan - macrofagen - receptoren - immunologie - visteelt - aquacultuur - carp - cyprinus - immune system - defence mechanisms - immune response - immunostimulation - beta-glucan - macrophages - receptors - immunology - fish culture - aquaculture

    Toll-like receptors (TLRs), named after the Toll gene identified in fruit flies, are a family of evolutionary conserved proteins that play a key role in the innate immune system. TLRs are found inside or on the surface of immune cells of virtually all-living animals and recognize integral parts of microbes. Thereby, they are excellent candidate receptors for controlled stimulation of the innate immune system of, for example, fish in aquaculture. β-glucans are microbial compounds routinely added to fish feed for their health-promoting effects. They regulate innate immunity by stimulating fish cells to produce more oxygen and nitrogen radicals but are not recognized by TLRs.Instead, TLRs of cyprinid fish (zebrafish, carp) are stimulated by viral and/or parasitic infection. Although immunostimulation by β-glucans occurs via yet undefined receptors certainly, addition of integral but harmless parts of microbes to fish feed may help controlfish diseases in aquaculture.

    Mathematical modelling of SERK mediated BR signalling
    Esse, G.W. van - \ 2013
    Wageningen University. Promotor(en): Sacco de Vries, co-promotor(en): Janwillem Borst; Simon van Mourik. - S.l. : s.n. - ISBN 9789461735553 - 263
    arabidopsis - planten - signaaltransductie - receptoren - brassinosteroïden - somatische embryogenese - modelleren - genexpressie - wiskundige modellen - arabidopsis - plants - signal transduction - receptors - brassinosteroids - somatic embryogenesis - modeling - gene expression - mathematical models

    Being sessile by nature plants are continuously challenged by biotic and abiotic stress factors. At the cellular level, different stimuli are perceived and translated to the desired response. In order to achieve this, signal transduction cascades have to be interlinked. Complex networks of downstream targets as well as positive and negative regulatory elements are essential for proper signal transduction. This often complicates analysis of signal transduction cascades via genetic approaches as a mutation in one gene results in a pleiotropic phenotype. Pathway components can be placed in the signal transduction cascade based on genetics as well as biochemical interactions between proteins. This results in a signal transduction network which is based on Boolean logics; either the gene is there and is functional (on) or it’s mutated and not functional (off). In such a genetic scheme, intermediate conditions and the effect of concentration on pathway components is not taken into account. Also, the effect of intermediate or transient activation states on signal transduction pathways is rarely included. In principle all proteins in a signal transduction network obey mass action laws suggesting that reaction rate and as a consequence the output of the signal depends on the concentration of the reactants. In addition, signals can be subjected to negative feedback thereby resulting in a signal that attenuates itself to maintain cellular homeostasis, or only responds to a stimulus temporarily and only when required. At the cellular level, the cell has to decipher all stimuli to enable the desired integrated cellular response. In this respect, concentration or amplitudes matter very much as the plant must not respond to background noise. Hence a cellular response will only be induced when a signal is above a certain threshold resulting in “switch like” behaviour of the system. Mathematical modelling can help to visualise and explain the temporal and concentration effects of pathway components on the output of signal transduction cascades. In order to do so, the signal transduction cascade needs to be well described with a clear and measurable response. Obviously, to know how receptor concentration affects the signalling output one has to know its numbers, and this was the starting point of the work described in this thesis. The final challenge was to describe the modulatory effect of SERK co-receptors on BR signalling. For this, SERK mediated BRI1 signalling was incorporated in a mathematical model that describes root growth and hypocotyl elongation based on the BRI1 receptor activity.

    In Chapter 1 the brassinosteroid signalling pathway as well as the role of SERK co-receptors on BRI1 mediated signalling is described. BRs are perceived by the plasma membrane localised Brassinosteroid insensitive 1 (BRI1) receptor. For its signalling, BRI1 completely depends on the presence of non-ligand binding co-receptors of the somatic embryogenesis receptor like kinase (SERK) co-receptor family. An added complexity is that SERK co-receptors associate with different main ligand perceiving receptors thereby affecting multiple signalling pathways simultaneously. Therefore, it is important to know how SERK co-receptors modulate the output of the main ligand perceiving receptor and how SERK co-receptors are distributed between the signal transduction cascades. The BRI1 signal transduction pathway is one of the best understood signal transduction cascades in Arabidopsis with clearly described ligands and associated phenotypes. For this reason, the focus of this study was on how SERK co-receptors affect BRI1 mediated signalling quantitatively using a mathematical modelling approach. This requires knowledge on the concentration of the main ligand perceiving receptor, SERK co-receptor and ligand levels. Since the BRI1 and SERK co-receptor concentration was unknown we set out to quantify the number of receptors in a cell. In Chapter 2 a confocal microscopy based method is described that enables quantification of BRI1, SERK1 and SERK3 in planta. The number of BRI1 receptor molecules in root epidermal cells ranges from 22,000 in the meristem to 80,000 in the maturation zone. However, when taking into account differences in cell size, the root meristem cells have the same receptor density which reduces significantly in the maturation zone. The root meristem cells are thought to be most active in BR signalling, suggesting that receptor density rather than total number of BRI1 receptors affects the sensitivity of a cell for BRs.

    The next question is, how the physiological response of the cell depends on both ligand stimulation of the receptor and on ligand concentration. To address this, a mathematical modelling approach was employed where the receptor - ligand concentrations were coupled to root growth and hypocotyl elongation as a downstream physiological readout for BR signalling (Chapter 3). Based on the BRI1 receptor activity the model faithfully predicts root growth as observed in bri1 loss-of-function mutants. The model also predicts that a rather low number of receptor molecules are needed to initiate a physiological response. Interestingly, the “switch” between activation and inhibition of root growth depends on the BRI1 occupancy level. This suggests that BRI1 may be a core regulator based on activating different targets based on its occupancy level. Root growth is robust against reduction in the BRI1 receptor level but not to variation in the BR concentration. This indicates that BR signalling is mainly regulated via ligand availability and biochemical activity. Since BRI1 signalling is highly dependent on the presence of SERK co-receptors, it is important to determine how these co-receptors affect the signalling output. Therefore, in Chapter 4, the BRI1 receptor model was extended with two co-receptors, SERK1 and SERK3. The model also takes into account BRI1 signalling independent of SERK1 and SERK3. This may occur due the activity of BRI1 alone, or due to interaction of BRI1 with another co-receptor, for example SERK4. It appears that roots of the serk1serk3 double mutant are almost completely irresponsive for BRs while the hypocotyl is not, suggesting either a difference in co-receptor usage or a higher activity of BRI1 alone in the hypocotyl. The usage of different co-receptors may reflect a mechanism by which the sensitivity of a cell for BRs is regulated. It appears that co-receptors mainly act by increasing the magnitude of the response. In addition, in silico simulations confirm that BRI1 signalling is not impaired when the majority of SERK co-receptors operate in other signalling pathways. The presented model provides a starting point to incorporate the effect of other modulators of the BRI1 signal transduction cascade on a complex physiological response.

    Current models for BRI1 mediated signalling postulate that SERK3 is recruited upon ligand binding. However, Fluorescence Recovery After Photo bleaching (FRAP) measurements described in Chapter 5, indicate that BRI1 receptors located in root meristem cells have a relatively low mobility. This suggests that BRI1 and SERK already form complexes in the absence of ligand.

    It has been repeatedly reported that SERK co-receptors are involved in various biological processes and signal transduction networks. In Chapter 6, the changes in gene expression in absence of functional SERK1 and SERK3 are studied using transcriptional analysis. Microarrays were performed on RNA isolated from roots of 4-day-old seedlings of serk1, serk3 and serk1serk3 mutants.

    Hierarchical cluster analysis indicated that serk3 mutant roots have the same transcriptional pattern when compared to roots of the serk1serk3 double mutant but to a lower magnitude. More than half of the genes differentially regulated in the serk1serk3 double mutant relate to BRI1 mediated signalling. In addition, a number of BR dependent and independent metabolic processes are affected in absence of SERK3 indicating that this co-receptor may have an additional function in metabolic control. Performing microarray analysis on receptor mutants is complicated as effects on gene transcripts may be indirect and due to differential regulation of downstream transcriptional regulators. This complexity is further enhanced in the SERK co-receptor mutants as multiple signalling pathways are affected. This raises the question if it is truly possible to correlate alterations in gene expression due to the absence of functional SERK co-receptors to one particular signal transduction pathway. In Chapter 7, the general discussion, it is described how modelling of BRI1 signalling in this thesis has contributed to new insights into the brassinosteroid signalling. Microscopy has been an important tool to quantify the number of receptors in a cell or the number of cells in a tissue. What is still needed is a clear link between a signalling activity, and, therefore, the physiological response of the cell, to local and intracellular protein-protein interactions and protein concentrations. Further expanding the available microscopic techniques and mathematical models to the cellular level is one of the next challenges. The research described in this thesis is a starting point for such an approach to study signal transduction in Arabidopsis.

    A low-density DNA microchip for the detection of (anti-)estrogenic compounds and their relative potencies
    Wang, S. ; Rijk, J.C.W. ; Pen, M.J. ; Aarts, J.M. ; Peijnenburg, A.A.C.M. ; Rietjens, I. ; Bovee, T.F.H. - \ 2013
    Analytical Biochemistry 435 (2013)1. - ISSN 0003-2697 - p. 83 - 92.
    i gene-expression - disrupting chemicals - breast-cancer - growth-factor - estrogen - receptors - protein - assays - alpha - identification
    In the current study, a set of 12 reference compounds was tested in a low-density DNA microchip that contains probes for 11 different estrogen-responsive marker genes. Our results show that the seven most informative marker genes on the chip resulted in fingerprints that correctly predicted the (anti-)estrogenic activity of the model compounds except that of the negative control testosterone. Two marker genes, myeloid leukemia factor-1 interacting protein and ubiquitin-conjugating enzyme E2C, were even capable of correctly predicting the estrogenic potency of all five estrogen receptor (ER) agonists tested and correlated well with the potencies as determined in the MCF-7/BOS proliferation assay and the in vivo uterotrophic assay. In addition, it was demonstrated that the estrogenic responses of testosterone, both in the array tube assay and in the proliferation assay, were partially due to the conversion of testosterone into 17ß-estradiol by aromatase but also due to formation of other estrogenic metabolites, the presence and estrogenic potency of which were confirmed by gas chromatography–tandem mass spectrometry analysis and a yeast-based reporter gene assay, respectively. It is concluded that low-density DNA microchip-based fingerprinting in MCF-7/BOS cells for estrogenicity marker genes provides a faster in vitro alternative to the current MCF-7/BOS cell proliferation assay (E-screen)
    On the modulation of innate immunity by plant-parasitic cyst nematodes
    Postma, W.J. - \ 2013
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - S.l. : s.n. - ISBN 9789461735560 - 154
    plantenparasitaire nematoden - globodera rostochiensis - heterodera schachtii - planten - interacties - immuniteit - immuunsysteem - modulatie - receptoren - signaaltransductie - moleculaire plantenziektekunde - plant parasitic nematodes - globodera rostochiensis - heterodera schachtii - plants - interactions - immunity - immune system - modulation - receptors - signal transduction - molecular plant pathology

    Plant-parasitic cyst nematodes are major agricultural pests worldwide. These obligate endoparasites invade the roots of host plants where they transform cells near the vascular cylinder into a permanent feeding site. Plants possess a multilayered innate immune system consisting of different types of extracellular and intracellular immune receptors. These enable detection of most invading nematodes and initiate immune responses that result in resistance. Many plant pathogens use effectors to overcome resistance. Here, modulation of plant innate immunity by plant-parasitic cyst nematodes was investigated. Extracellular immune receptor signaling and hormone-mediated signaling pathways were found to contain infection of susceptible Arabidopsis thalianawith Heterodera schachtii. A large family of effectors was identified in Globodera rostochiensis. One of these so-called SPRYSECs interacted with a novel CC-NB-LRR type resistance protein of a susceptible tomato without inducing resistance responses. Instead, the effector was found to suppress defense-related programmed cell death and resistance mediated by several CC-NB-LRR type resistance proteins. In addition, a secreted antimicrobial peptide was identified in G. rostochiensis. Plant-parasitic cyst nematodes thus most likely secrete effectors that protect against plant immune responses and secondary infections. The current evidence for the existence of immune modulating effectors is reviewed and directions for further research are given.

    Functional analysis of tomato immune receptor Ve1 and recognition of Verticillium effector Ave1
    Zhang, Z. - \ 2013
    Wageningen University. Promotor(en): Bart Thomma; Pierre de Wit, co-promotor(en): C.M. Liu. - S.l. : s.n. - ISBN 9789461735461 - 191
    solanum lycopersicum - tomaten - celwanden - receptoren - immuunsysteem - liganden - plantenziekteverwekkende schimmels - verticillium - infectiviteit - modellen - plant-microbe interacties - solanum lycopersicum - tomatoes - cell walls - receptors - immune system - ligands - plant pathogenic fungi - verticillium - infectivity - models - plant-microbe interactions

    Similar to the animal innate immune system, plants employ extracellular leucine rich repeat (eLRR)-containing cell surface receptors to recognize conserved molecular structures that are derived from microbial pathogens. A number of these immune receptors, as well as the corresponding pathogen ligands, have been characterized. The interaction between the tomato Ve1 immune receptor and the Ave1 effector from the pathogenic fungus Verticillium serves as a model system for the study of plant innate immunity. The research described in this thesis was aimed at a further understanding of how the eLRR-containing cell surface receptor Ve1 confers recognition of the Ave1 ligand and how it activates downstream immune signaling.

    It has been shown that eLRR-containing cell surface receptors play important roles in development and innate immunity in various plant species.Chapter 1 gives an overview on the current status of research on eLRR-containing cell surface receptors, their co-receptors and corresponding ligands, with emphasis on structural aspects. The functions of distinct eLRR receptor domains, their role in structural conformation, ligand perception, signal transduction and receptor complex formation are extensively discussed.

    To facilitate studies on the Ve1-Ave1 model system, we describe the establishment of protocols to investigate Ve1-mediated recognition of Ave1 and immune signaling in tobacco in Chapter 2. We optimized an Agrobacterium tumefaciens transient expression assay (agroinfiltration) by testing various over-expression vectors, and found that co-expression of Ve1 and Ave1 leads to hypersensitive response (HR) only in particular tobacco species. We further report on virus-induced gene silencing (VIGS) in Nicotiana tabacum cv. Samsun that allows investigating signaling components involved in Ve1-mediated resistance. Collectively, we established N. tabacum as a model plant to study Ve1-mediated immunity.

    In Chapter 3, we further investigated whether co-expression of Ve1 and Ave1 in Arabidopsis leads to an HR, which may potentially be used as a straightforward screening method upon a random mutagenesis. However, although Ave1 is able to trigger an HR in resistant tomato and tobacco plants, co-expression of Ve1 and Ave1 did not activate an HR in Arabidopsis. These results suggest that the HR occurs as a consequence of Ve1-mediated resistance signaling, and it is not absolutely required for Verticillium resistance.

    In Chapter 4 we investigated the contribution of particular regions of Ve1 to the activation of immune signaling through domain swaps between Ve1 with its non-functional homolog Ve2. Agroinfiltration, as well as stable Arabidopsis transformation, revealed that chimeras in which the first thirty eLRRs of Ve1 were replaced with those of Ve2 remain able to induce HR and activate Verticillium resistance. However, a truncated Ve1 protein that lacks the first 30 eLRRs is no longer functional. We speculate that the non-functional Ve2 receptor may still interact with the Ave1 effector in the eLRR domain, but fails to activate immune signaling due to a non-functional C-terminus.

    In Chapter 5, site-directed mutagenesis was employed to further investigate the eLRR domain of Ve1. We designed alanine scanning mutants in the solvent-exposed residues across the convex surface of the eLRR domain. In each mutant, two of the five solvent-exposed residues in β-sheet of a single eLRR were substituted into alanines. Functionality of the mutants through agroinfiltration and stable transformation of Arabidopsis revealed three eLRR regions that are potentially required for ligand specificity and for co-receptor interaction. In addition, alanine substitution was employed to evaluate role of putative protein-protein interaction and endocytosis motifs in the transmembrane domain and the cytoplasmic tail of the Ve1 protein. However, no requirement of these domains for Ve1 functionality could be demonstrated.

    It has been demonstrated that eLRR-containing cell-surface immune receptors often recognize short peptide sequence stretches as epitopes of their ligands. In Chapter 6, we aimed to identify the surface epitope of the Verticilliumeffector Ave1 that is recognized by Ve1. Firstly, we assessed whether various Ave1 homologs are recognized by Ve1. Since we found that C-terminal fusion of a GFP tag to Ave1 compromised its recognition, we hypothesized that accessibility of the Ave1 C-terminus is essential for Ve1-mediated recognition. Ave1 truncations and domain swaps with Ave1 homologs that are not recognized by Ve1 showed that a nine amino acid sequence derived from the C-terminus of Ave1 is essential for recognition by Ve1. This nine amino acid epitope is sufficient to activate Ve1-mediated immunity.

    In Chapter 7 the highlights of the thesis are discussed and placed in a broader perspective. The current understanding of eLRR-containing cell surface receptors is discussed, taking the findings of this thesis into account, with specific emphasis on ligand perception and receptor complex formation. In addition, future perspectives on the future are sketched, and novel research questions are posed aimed to obtain further insights into how Ve1 may form complexes with various co-receptors and how Ave1 contributes to Verticillium pathogenicity.

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