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.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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CRISPR/Cas inactivation of RECQ4 increases homeologous crossovers in an interspecific tomato hybrid
Maagd, Ruud A. de; Loonen, Annelies ; Chouaref, Jihed ; Pelé, Alexandre ; Meijer-Dekens, Fien ; Fransz, Paul ; Bai, Yuling - \ 2019
Plant Biotechnology Journal (2019). - ISSN 1467-7644
class II crossover pathway - interspecific crosses - introgression breeding - meiosis - RECQ4

Crossover formation during meiosis in plants is required for proper chromosome segregation and is essential for crop breeding as it allows an (optimal) combination of traits by mixing parental alleles on each chromosome. Crossover formation commences with the production of a large number of DNA double-strand breaks, of which only a few result in crossovers. A small number of genes, which drive the resolution of DNA crossover intermediate structures towards non-crossovers, have been identified in Arabidopisis thaliana. In order to explore the potential of modification of these genes in interspecific hybrids between crops and their wild relatives towards increased production of crossovers, we have used CRISPR/Cas9-mutagenesis in an interspecific tomato hybrid to knockout RecQ4. A biallelic recq4 mutant was obtained in the F1 hybrid of Solanum lycopersicum and S. pimpinellifolium. Compared with the wild-type F1 hybrid, the F1 recq4 mutant was shown to have a significant increase in crossovers: a 1.53-fold increase when directly observing ring bivalents in male meiocytes microscopically and a 1.8-fold extension of the genetic map when measured by analysing SNP markers in the progeny (F2) plants. This is one of the first demonstrations of increasing crossover frequency in interspecific hybrids by manipulating genes in crossover intermediate resolution pathways and the first to do so by directed mutagenesis. Significance statement: Increasing crossover frequency during meiosis can speed up or simplify crop breeding that relies on meiotic crossovers to introduce favourable alleles controlling important traits from wild relatives into crops. Here we show for the first time that knocking out an inhibitor of crossovers in an interspecific hybrid between tomato and its relative wild species using CRISPR/Cas9-mutagenesis results in increased recombination between the two genomes.

Development of equine intestinal organoid monolayers to study location-specific epithelial responses
Hee, Bart van der; Doorn, David van; Loonen, L.M.P. ; Moolen, Kitty van der; Smidt, H. ; Wells, J.M. - \ 2019
Employing the self-replicative potential of stem cells to generate equine mini guts
0046 - Organoids as models to study probiotics
Hee, B. van der; Loonen, L.M.P. ; Taverne, N. ; Taverne-Thiele, J.J. ; Smidt, H. ; Wells, J.M. - \ 2019
- 1 p.
permeability - intestinal models - organoids - stem-cells - tight-junctions
For decades, scientists have exploited cancer cell lines as models to study host-pathogen interactions and intestinal functions in vitro. Such monotypic cell models have led to important discoveries but have notable limitations. Immortalized cell lines display biological variations such as aneuploidy, chromosome rearrangements or mutations leading to poorly reproducible results, even for the same cell line.
Organoids are gaining considerable interest as alternative models of the intestine due to their close resemblance to structural, cellular and functional complexity found in vivo. However, the three-dimensional geometry of stem-cell derived intestinal organoids limits easy access to the apical epithelium for investigating the influence of probiotics, bioactive and toxic compounds on barrier function and permeability. Here we present a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated organoids. Confluent polarised monolayers containing tight-junctions were formed in three days and could be used in experiments for up to two weeks. Multilineage differentiation of the ileal stem cells was demonstrated by immunohistochemistry, and RT-qPCR of cell-specific transcripts. Furthermore, we showed that adult stem-cell derived ileal organoids maintain location-specific transcriptional programs during long-term in vitro culture.
Development of equine intestinal organoid monolayers to study location-specific epithelial responses : Employing the self-replicative potential of stem cells to generate equine mini guts
Hee, B. van der; Doorn, David van; Loonen, L.M.P. ; Moolen, Kitty van der; Smidt, H. ; Wells, J.M. - \ 2019
Equine - Organoids - Monolayer - equine - organoids - monolayer - epithelium - cecum - jejunum - colon - Host-microbe interactions
Polyphenols and Tryptophan Metabolites Activate the Aryl Hydrocarbon Receptor in an in vitro Model of Colonic Fermentation
Koper, Jonna E.B. ; Loonen, Linda M.P. ; Wells, Jerry M. ; Troise, Antonio Dario ; Capuano, Edoardo ; Fogliano, Vincenzo - \ 2019
Molecular Nutrition & Food Research 63 (2019)3. - ISSN 1613-4125
aryl hydrocarbon receptor - luteolin - microbiota - SHIME - tryptophan

Scope: Many dietary phytochemicals have been reported to promote gut health. Specific dietary phytochemicals, such as luteolin, as well as specific microbial metabolites of tryptophan are ligands of the aryl hydrocarbon receptor (AhR), which plays a role in immunity and homeostasis of the gut barrier. Here, the fate of luteolin during colonic fermentation and the contribution of tryptophan metabolites to AhR activity in different parts of the colon are investigated. Methods and results: Several polyphenols are screened for AhR activation and oregano, containing the ligand luteolin, is added to batch cultures of human microbiota from the distal colon. Luteolin is rapidly metabolized, with no measurable increase in AhR activity. In the second experiment, using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME), not all luteolin is metabolized in the ascending colon, but disappear rapidly in the transverse colon. The greatest AhR activity is due to microbiota-derived metabolites of tryptophan, particularly in the descending colon. Conclusions: Luteolin in food is rapidly metabolized in the transverse colon. Tryptophan metabolism by the microbiota in the colon contributes substantially to the pool of lumen metabolites that can activate the AhR.

Stress behaviour and physiology of developing Arctic barnacle goslings (Branta leucopsis) is affected by legacy trace contaminants
Scheiber, Isabella B.R. ; Weiß, Brigitte M. ; Jong, Margje E. De; Braun, Anna ; Brink, Nico W. Van Den; Loonen, Maarten J.J.E. ; Millesi, Eva ; Komdeur, Jan - \ 2018
Proceedings of the Royal Society. B: Biological Sciences 285 (2018)1893. - ISSN 0962-8452
acute stress behaviour - Arctic - barnacle goose (Branta leucopsis) - HPA corticosterone metabolites - legacy trace metal contamination - stress coping

Natural populations are persistently exposed to environmental pollution, which may adversely impact animal physiology and behaviour and even compromise survival. Responding appropriately to any stressor ultimately might tip the scales for survival, as mistimed behaviour and inadequate physiological responses may be detrimental. Yet effects of legacy contamination on immediate physiological and behavioural stress coping abilities during acute stress are virtually unknown. Here, we assessed these effects in barnacle goslings (Branta leucopsis) at a historical coal mine site in the Arctic. For three weeks we led human-imprinted goslings, collected from nests in unpolluted areas, to feed in an abandoned coal mining area, where they were exposed to trace metals. As control we led their siblings to feed on clean grounds. After submitting both groups to three well-established stress tests (group isolation, individual isolation, on-back restraint), control goslings behaved calmer and excreted lower levels of corticosterone metabolites. Thus, legacy contamination may decisively change stress physiology and behaviour in long-lived vertebrates exposed at a young age.

Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids
Hee, B. van der; Loonen, L.M.P. ; Taverne, N. ; Taverne-Thiele, J.J. ; Smidt, H. ; Wells, J.M. - \ 2018
Stem Cell Research 28 (2018). - ISSN 1873-5061 - p. 165 - 171.
Epithelium - Intestinal - Monolayer - Organoids - Porcine
An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology.
Mercury associated neurochemical response in Arctic barnacle goslings (Branta leucopsis)
Brink, Nico W. van den; Scheiber, Isabella B.R. ; Jong, Margje E. de; Braun, Anna ; Arini, Adeline ; Basu, Niladri ; Berg, Hans van den; Komdeur, Jan ; Loonen, Maarten J.J.E. - \ 2018
Science of the Total Environment 624 (2018). - ISSN 0048-9697 - p. 1052 - 1058.
Exposure and effect - Neurotoxicity - Polar - Terrestrial - Tundra
There remains great concern over mercury pollution in the Arctic, though relatively little is known about impacts on biota that inhabit Arctic terrestrial systems. To help address this, the current study was performed with barnacle goslings (Branta leucopsis) from a coal mine-impacted site and a control site near Ny-Ålesund, Spitsbergen (Svalbard). The works focused mainly on mercury, as coal contains trace levels of this element. Total mercury concentrations were quantified in soil and vegetation from the two sites, as well as feces and liver from the goslings. Next, the mercury exposures were related to dopamine 2 (D2)- and NMDA-receptors in the brain, given that mercury is a proven neurotoxicant. Soil and vegetation in the mining area contained mercury levels that were approximately 3- and 2.2-times higher than in the control site. Despite a significant difference between the sites, the soil and vegetation mercury levels where were within ranges found at other Arctic locations. Goslings grazing in the mine-impacted area contained significantly higher hepatic mercury levels than those sampled from the control site. Compared to other species, the hepatic concentrations were relatively low possibly due to dilution of the mercury in growing goslings (growth dilution) and deposition of mercury in the growing feathers. Hepatic mercury concentrations were positively related to D2-neuroreceptor levels but not to NMDA-receptor levels thus suggesting a possible subtle neurological effect. To our knowledge, this is among the first studies on mercury exposure in Arctic terrestrial organisms, and one of the first to document potential subtle neurological responses associated with exposure to low, environmentally relevant mercury levels, which also can be found at other locations in the Arctic. However, as a pilot effort, the results here need to be examined in additional studies that include, for example, lager study designs, different geographic sites and other terrestrial species.
Indices of stress and immune function in Arctic barnacle goslings (Branta leucopsis) were impacted by social isolation but not a contaminated grazing environment
Jong, Margje E. de; Scheiber, Isabella B.R. ; Brink, Nico W. van den; Braun, Anna ; Matson, Kevin D. ; Komdeur, Jan ; Loonen, Maarten J.J.E. - \ 2017
Science of the Total Environment 601-602 (2017). - ISSN 0048-9697 - p. 132 - 141.
Acute phase proteins - Complement - Corticosterone - Heavy metals - Natural antibodies - Nitric oxide

In many areas around the Arctic remains and spoil heaps of old mines can be found, which have been abandoned after their heydays. Runoff from tailings of these abandoned mines can directly contaminate the local environment with elevated concentrations of trace metals. Few studies have investigated the possible negative effects of contaminants on Arctic terrestrial animals that use these areas. Trace metals can accumulate in animals and this accumulation has been linked to negative effects on fitness. Both, the hypothalamus-pituitary-adrenal (HPA) axis and/or the immune system have been named as possible underlying causes for these observations. Free-living animals are often exposed to multiple stressors simultaneously, however, and this is often not considered in studies on the effects of contaminants on animal physiology. Here, we performed a study on Spitsbergen (Svalbard) taking both potential effects of trace metal contamination and social stress into account. We investigated experimentally effects of exposure to contaminants from a historic coal mine area on plasma corticosterone levels and on four innate immune parameters (haemolysis, haemagglutination, haptoglobin-like activity and nitric oxide) before and after social isolation in human-raised barnacle goslings (Branta leucopsis). Baseline corticosterone and immune parameters were not affected by mine-exposure. After social isolation, mine goslings tended to show decreased haemagglutination in comparison with control goslings, but we detected no difference in the other measures. Social isolation increased corticosterone and decreased haptoglobin-like activity in all goslings. Immunology and corticosterone levels of barnacle goslings thus seem unaffected, at least on the short term, by Arctic coal mining contamination.

Balancing ecosystem function, services and disservices resulting from expanding goose populations
Buij, Ralph ; Melman, Theodorus C.P. ; Loonen, Maarten J.J.E. ; Fox, Anthony D. - \ 2017
Ambio 46 (2017). - ISSN 0044-7447 - p. 301 - 318.
Ecosystem functions - Ecosystem services - Goose overabundance - Herbivores - Species interactions
As goose populations increase in abundance, their influence on ecological processes is increasing. We review the evidence for key ecological functions of wild goose populations in Eurasia and North America, including aquatic invertebrate and plant propagule transport, nutrient deposition in terrestrial and aquatic ecosystems, the influence of goose populations on vegetation biomass, carbon storage and methane emission, species diversity and disease transmission. To estimate the implications of their growing abundance for humans, we explore how these functions contribute to the provision of ecosystem services and disservices. We assess the weight, extent and trends among such impacts, as well as the balance of their value to society. We examine key unresolved issues to enable a more balanced assessment of the economic costs or benefits of migratory geese along their flyways, including the spatial and temporal variation in services and their contrasting value to different user groups. Many ecological functions of geese are concluded to provide neither services nor disservices and, ecosystem disservices currently appear to outweigh services, although this varies between regions. We consider an improved quantification of ecosystem services and disservices, and how these vary along population flyways with respect to variation in valuing certain cultural services, and under different management scenarios aimed at reducing their disservices, essential for a more balanced management of goose populations.
Silencing of six susceptibility genes results in potato late blight resistance
Sun, Kaile ; Wolters, Anne-Marie A. ; Vossen, Jack H. ; Rouwet, Maarten E. ; Loonen, Annelies E.H.M. ; Jacobsen, Evert ; Visser, Richard G.F. ; Bai, Yuling - \ 2016
Transgenic Research 25 (2016)5. - ISSN 0962-8819 - p. 731 - 742.
Late blight - Potato - Resistance - RNAi - Susceptibility gene

Phytophthora infestans, the causal agent of late blight, is a major threat to commercial potato production worldwide. Significant costs are required for crop protection to secure yield. Many dominant genes for resistance (R-genes) to potato late blight have been identified, and some of these R-genes have been applied in potato breeding. However, the P. infestans population rapidly accumulates new virulent strains that render R-genes ineffective. Here we introduce a new class of resistance which is based on the loss-of-function of a susceptibility gene (S-gene) encoding a product exploited by pathogens during infection and colonization. Impaired S-genes primarily result in recessive resistance traits in contrast to recognition-based resistance that is governed by dominant R-genes. In Arabidopsis thaliana, many S-genes have been detected in screens of mutant populations. In the present study, we selected 11 A. thalianaS-genes and silenced orthologous genes in the potato cultivar Desiree, which is highly susceptible to late blight. The silencing of five genes resulted in complete resistance to the P. infestans isolate Pic99189, and the silencing of a sixth S-gene resulted in reduced susceptibility. The application of S-genes to potato breeding for resistance to late blight is further discussed.

Identification of commensal species positively correlated with early stress responses to a compromised mucus barrier
Sovran, Bruno ; Lu, Peng ; Loonen, Linda M.P. ; Hugenholtz, Floor ; Belzer, Clara ; Stolte, Ellen H. ; Boekschoten, Mark V. ; Baarlen, Peter Van; Smidt, Hauke ; Kleerebezem, Michiel ; Vos, Paul De; Renes, Ingrid B. ; Wells, Jerry M. ; Dekker, Jan - \ 2016
Inflammatory Bowel Diseases 22 (2016)4. - ISSN 1078-0998 - p. 826 - 840.
Bacteroidetes - colitis - Muc2 deficiency - stress markers

Background: Our aims were (1) to correlate changes in the microbiota to intestinal gene expression before and during the development of colitis in Muc2 -/- mice and (2) to investigate whether the heterozygote Muc2 +/- mouse would reveal host markers of gut barrier stress. Methods: Colon histology, transcriptomics, and microbiota profiling of faecal samples was performed on wild type, Muc2 +/-, and Muc2 -/- mice at 2, 4, and 8 weeks of age. Results: Muc2 -/- mice develop colitis in proximal colon after weaning, resulting in inflammatory and adaptive immune responses, and expression of genes associated with human inflammatory bowel disease. Muc2 +/- mice do not develop colitis, but produce a thinner mucus layer. The transcriptome of Muc2 +/- mice revealed differential expression of genes participating in mucosal stress responses and exacerbation of a transient inflammatory state around the time of weaning. Young wild type and Muc2 +/- mice have a more constrained group of bacteria as compared with the Muc2 -/- mice, but at 8 weeks the microbiota composition is more similar in all mice. At all ages, microbiota composition discriminated the groups of mice according to their genotype. Specific bacterial clusters correlated with altered gene expression responses to stress and bacteria, before colitis development, including colitogenic members of the genus Bacteroides. Conclusions: The abundance of Bacteroides pathobionts increased before histological signs of pathology suggesting they may play a role in triggering the development of colitis. The Muc2 +/- mouse produces a thinner mucus layer and can be used to study mucus barrier stress in the absence of colitis.

Down-regulation of Arabidopsis DND1 orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew
Sun, K. ; Wolters, A.M.A. ; Loonen, A.E.H.M. ; Huibers, R.P. ; Vlugt, R.A.A. van der; Goverse, A. ; Jacobsen, E. ; Visser, R.G.F. ; Bai, Y. - \ 2016
Transgenic Research 25 (2016). - ISSN 0962-8819 - p. 123 - 138.
Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.
Mercury in the Arctic terrestrial Ecosystem effects of Hg on Barnacle goslings
Brink, N.W. van den; Jong, Margje de; Loonen, Maarten - \ 2015
In: An overview of Arctic research of Wageningen UR Wageningen UR - 1 p.
IL-22-STAT3 Pathway Plays a Key Role in the Maintenance of Ileal Homeostasis in Mice Lacking Secreted Mucus Barrier
Sovran, B. ; Loonen, L.M.P. ; Hugenholtz, F. ; Belzer, C. ; Kranenbarg-Stolte, H.H. ; Boekschoten, M.V. ; Baarlen, P. van; Kleerebezem, M. ; Vos, P. de; Dekker, J. ; Renes, I.B. ; Wells, J.M. - \ 2015
Inflammatory Bowel Diseases 21 (2015)3. - ISSN 1078-0998 - p. 531 - 542.
inflammatory-bowel-disease - pancreatitis-associated protein - intestinal epithelial-cells - colonic gene-expression - experimental colitis - muc2-deficient mice - mucin muc2 - microbiota - bacteria - stat3
Background: Muc2-deficient mice show no signs of ileal pathology but the mechanisms remained unknown. Methods: Wild-type (WT), Muc2+/-, and Muc2-/- mice were killed at 2, 4, and 8 weeks of age. Total RNA from ileum was used for full genome transcriptome analysis and qPCR. Microbiota composition was determined using a mouse intestinal chip (MITChip). Morphological and immunohistological studies were performed on segments of ileum. Results: The ileum was colonized by more diverse microbiota in young (week 4) WT than in Muc2-/- mice, and composition was influenced by genotype. Weaning was associated with major changes in the transcriptome of all mice, and the highest number of differentially expressed genes compared with adults, reflecting temporal changes in microbiota. Although the spatial compartmentalization of bacteria was compromised in Muc2-/- mice, gene set enrichment analysis revealed a downregulation of Toll-like receptor, immune, and chemokine signaling pathways compared to WT mice. The predicted effects of enhanced IL-22 signaling were identified in the Muc2-/- transcriptome as the upregulation of epithelial cell proliferation altered expression of mitosis and cell-cycle control pathways. This is consistent with increased villus length and number of Ki67+ epithelial cells in Muc2-/- mice. Additionally, expression of the network of IL-22 regulated defense genes, including Fut2, Reg3[beta], Reg3[gamma], Relmb, and the Defensin Defb46 were increased in Muc2-/- mice. Conclusions: These findings highlight a role for the IL-22-STAT3 pathway in maintaining ileal homeostasis when the mucus barrier is compromised and its potential as a target for novel therapeutic strategies in inflammatory bowel disease
Natural loss-of-function mutation of EDR1 conferring resistance to tomato powdery mildew in Arabidopsis thaliana accession C24
Gao, D. ; Appiano, M. ; Huibers, R.P. ; Loonen, A.E.H.M. ; Visser, R.G.F. ; Wolters, A.M.A. ; Bai, Y. - \ 2015
Molecular Plant Pathology 16 (2015)1. - ISSN 1464-6722 - p. 71 - 82.
salicylic-acid - downy mildew - gene - defense - plants - microsatellites - mechanism - evolution - cloning - kinase
To screen for potentially novel types of resistance to tomato powdery mildew Oidium neolycopersici, a disease assay was performed on 123 Arabidopsis thaliana accessions. Forty accessions were fully resistant, and one, C24, was analysed in detail. By quantitative trait locus (QTL) analysis of an F2 population derived from C24 × Sha (susceptible accession), two QTLs associated with resistance were identified in C24. Fine mapping of QTL-1 on chromosome 1 delimited the region to an interval of 58¿kb encompassing 15 candidate genes. One of these was Enhanced Disease Resistance 1 (EDR1). Evaluation of the previously obtained edr1 mutant of Arabidopsis accession Col-0, which was identified because of its resistance to powdery mildew Golovinomyces cichoracearum, showed that it also displayed resistance to O.¿neolycopersici. Sequencing of EDR1 in our C24 germplasm (referred to as C24-W) revealed two missing nucleotides in the second exon of EDR1 resulting in a premature stop codon. Remarkably, C24 obtained from other laboratories does not contain the EDR1 mutation. To verify the identity of C24-W, a DNA region containing a single nucleotide polymorphism (SNP) unique to C24 was sequenced showing that C24-W contains the C24-specific nucleotide. C24-W showed enhanced resistance to O.¿neolycopersici compared with C24 not containing the edr1 mutation. Furthermore, C24-W displayed a dwarf phenotype, which was not associated with the mutation in EDR1 and was not caused by the differential accumulation of pathogenesis-related genes. In conclusion, we identified a natural edr1 mutant in the background of C24.
REG3γ-deficient mice have altered mucus distribution and increased mucosal inflammatory responses to the microbiota and enteric pathogens in the ileum
Baarlen, Peter van; Loonen-Jaklofsky, Linda ; Wells, Jerry - \ 2014
GSE44409 - Mus musculus - PRJNA189965
Objective: Reg3g has been proposed to have a protective role against infection due to its bactericidal effect on Gram-positive bacteria, but evidence from in vivo studies is lacking. Therefore we generated a Reg3g-/- mouse, to determine its role in intestinal homeostasis and protection against experimental infection. Methods: Reg3g-/- mice were phenotyped using histological methods and a range of innate and immune markers. To investigate the antimicrobial role of Reg3g we experimentally infected mice with Gram-positive Listeria monocytogenes and Gram-negative Salmonella entertitidis and measured translocated bacteria, mucosal and systemic markers of infection. Results: Reg3g-/- mice display altered ileal mucus distribution and increased bacterial contact with the epithelium. , concomitant with This increased the inflammatory status in of the ileal mucosa and increased expression of Il-22, myeloperoxidase (MPO) and serum chemokines in serum. In response to infection, Reg3g-/- mice showed transcriptome changes and elevated levels of mucosal MPO in the ileum, but no increased bacterial translocation to the organs. Conclusions: Reg3g is equally distributed throughout the mucus of wild type (wt) mice and its absence results in an altered distribution of the ileal mucus. Reg3g deficiency also results resulted in increased bacterial contact with the epithelium and heightened inflammatory responses in the ileal mucosa. We propose that Reg3g binds pathogens suggesting it and contributes to mucus barrier function by ensnaring bacteria. Compared to wt mice, Reg3g-/- mice infected with S. enteritidis and L. monocytogenes show an increase of mucosal inflammatory markers indicating the protective, anti-microbial roles of Reg3g in defence against both Gram-positive and -negative bacteria.
Activation tagging of ATHB13 in Arabidopsis thaliana confers broad-spectrum disease resistance
Gao, D. ; Huibers, R.P. ; Chen, X. ; Loonen, A.E.H.M. ; Visser, R.G.F. ; Wolters, A.M.A. ; Bai, Y. - \ 2014
Plant Molecular Biology 86 (2014)6. - ISSN 0167-4412 - p. 641 - 653.
vegetative storage protein - powdery mildew resistance - transcription factors hahb1 - nudix hydrolase - salicylic-acid - plant defense - cell-death - hd-zip - oidium-neolycopersici - expression patterns
Powdery mildew species Oidium neolycopersici (On) can cause serious yield losses in tomato production worldwide. Besides on tomato, On is able to grow and reproduce on Arabidopsis. In this study we screened a collection of activation-tagged Arabidopsis mutants and identified one mutant, 3221, which displayed resistance to On, and in addition showed a reduced stature and serrated leaves. Additional disease tests demonstrated that the 3221 mutant exhibited resistance to downy mildew (Hyaloperonospora arabidopsidis) and green peach aphid (Myzus persicae), but retained susceptibility to bacterial pathogen Pseudomonas syringae pv tomato DC3000. The resistance trait and morphological alteration were mutually linked in 3221. Identification of the activation tag insertion site and microarray analysis revealed that ATHB13, a homeodomain-leucine zipper (HD-Zip) transcription factor, was constitutively overexpressed in 3221. Silencing of ATHB13 in 3221 resulted in the loss of both the morphological alteration and resistance, whereas overexpression of the cloned ATHB13 in Col-0 and Col-eds1-2 backgrounds resulted in morphological alteration and resistance. Microarray analysis further revealed that overexpression of ATHB13 influenced the expression of a large number of genes. Previously, it was reported that ATHB13-overexpressing lines conferred tolerance to abiotic stress. Together with our results, it appears that ATHB13 is involved in the crosstalk between abiotic and biotic stress resistance pathways.
Down-regulation of acetolactate synthase compromises OI-1- mediated resistance to powdery mildew in tomato
Gao, D. ; Huibers, R.P. ; Loonen, A.E.H.M. ; Visser, R.G.F. ; Wolters, A.M.A. ; Bai, Y. - \ 2014
BMC Plant Biology 14 (2014). - ISSN 1471-2229 - 11 p.
glutamate-dehydrogenase gene - acetohydroxyacid synthase - monogenic-resistance - defense responses - nicotiana-tabacum - ol-genes - arabidopsis - plants - inhibition - biosynthesis
Background - In a cDNA-AFLP analysis comparing transcript levels between powdery mildew (Oidium neolycopersici)-susceptible tomato cultivar Moneymaker (MM) and near isogenic lines (NILs) carrying resistance gene Ol-1 or Ol-4, a transcript-derived fragment (TDF) M11E69-195 was found to be present in NIL-Ol-1 but absent in MM and NIL-Ol-4. This TDF shows homology to acetolactate synthase (ALS). ALS is a key enzyme in the biosynthesis of branched-chain amino acids valine, leucine and isoleucine, and it is also a target of commercial herbicides. Results - Three ALS homologs ALS1, ALS2, ALS3 were identified in the tomato genome sequence. ALS1 and ALS2 show high similarity, whereas ALS3 is more divergent. Transient silencing of both ALS1 and ALS2 in NIL-Ol-1 by virus-induced gene silencing (VIGS) resulted in chlorotic leaf areas that showed increased susceptibility to O. neolycopersici (On). VIGS results were confirmed by stable transformation of NIL-Ol-1 using an RNAi construct targeting both ALS1 and ALS2. In contrast, silencing of the three ALS genes individually by RNAi constructs did not compromise the resistance of NIL-Ol-1. Application of the herbicide chlorsulfuron to NIL-Ol-1 mimicked the VIGS phenotype and caused loss of its resistance to On. Susceptible MM and On-resistant line NIL-Ol-4 carrying a nucleotide binding site and leucine rich repeat (NB-LRR) resistance gene were also treated with chlorsulfuron. Neither the susceptibility of MM nor the resistance of NIL-Ol-4 was affected. Conclusions - ALS is neither involved in basal defense, nor in resistance conferred by NB-LRR type resistance genes. Instead, it is specifically involved in Ol-1-mediated resistance to tomato powdery mildew, suggesting that ALS-induced change in amino acid homeostasis is important for resistance conferred by Ol-1.
REG3¿-deficient mice have altered mucus distribution and increased mucosal inflammatory responses to the microbiota and enteric pathogens in the ileum
Loonen, L.M.P. ; Kranenbarg-Stolte, H.H. ; Jaklofsky, M.T.J. ; Meijerink, M. ; Dekker, J. ; Baarlen, P. van; Wells, J. - \ 2014
Mucosal Immunology 7 (2014). - ISSN 1933-0219 - p. 939 - 947.
pancreatitis-associated protein - bacterial aggregation - dietary calcium - bowel-disease - regiii-gamma - expression - salmonella - infection - lectin - rats
REG3¿ is considered to have a protective role against infection with Gram-positive bacteria due to its bactericidal activity, but evidence from in vivo studies is lacking. We generated a REG3¿-/- mouse, and investigated the effect of lack of REG3¿ on intestinal mucus distribution, spatial compartmentalization of bacteria, and expression of innate immunity genes. Infection studies were also performed with Gram-positive and Gram-negative pathogens to investigate the antimicrobial role of REG3¿. REG3¿-/- mice display altered mucus distribution, increased bacterial contact with the epithelium, and elevated inflammatory markers in the ileum without histological evidence of pathology. Infection response pathway genes were differentially expressed in both Listeria monocytogenes and Salmonella enteritidis infected REG3¿-/- and wild-type (wt) mice. Higher amounts of myeloperoxidase and interleukin-22 transcripts were present in the ileal mucosa of REG3¿-/- than wt mice, but translocation to the organs was unaffected. We concluded that REG3¿ has a protective role against mucosal infection with pathogenic Listeria and Salmonella in vivo. REG3¿ is equally distributed throughout the mucus and its absence results in increased epithelial contact with the microbiota resulting in low-grade inflammation. REG3¿ can bind to Gram-negative and Gram-positive bacteria and influence mucus distribution in the ileum, properties which may contribute to mucosal protection.
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