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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    Natural variation in HsfA2 pre-mRNA splicing is associated with changes in thermotolerance during tomato domestication
    Hu, Yangjie ; Mesihovic, Anida ; Jiménez-Gómez, José M. ; Röth, Sascha ; Gebhardt, Philipp ; Bublak, Daniela ; Bovy, Arnaud ; Scharf, Klaus Dieter ; Schleiff, Enrico ; Fragkostefanakis, Sotirios - \ 2020
    New Phytologist 225 (2020)3. - ISSN 0028-646X - p. 1297 - 1310.
    acclimation - high temperature - polymorphism - pre-mRNA splicing - Solanum - stress response

    Wild relatives of crops thrive in habitats where environmental conditions can be restrictive for productivity and survival of cultivated species. The genetic basis of this variability, particularly for tolerance to high temperatures, is not well understood. We examined the capacity of wild and cultivated accessions to acclimate to rapid temperature elevations that cause heat stress (HS). We investigated genotypic variation in thermotolerance of seedlings of wild and cultivated accessions. The contribution of polymorphisms associated with thermotolerance variation was examined regarding alterations in function of the identified gene. We show that tomato germplasm underwent a progressive loss of acclimation to strong temperature elevations. Sensitivity is associated with intronic polymorphisms in the HS transcription factor HsfA2 which affect the splicing efficiency of its pre-mRNA. Intron splicing in wild species results in increased synthesis of isoform HsfA2-II, implicated in the early stress response, at the expense of HsfA2-I which is involved in establishing short-term acclimation and thermotolerance. We propose that the selection for modern HsfA2 haplotypes reduced the ability of cultivated tomatoes to rapidly acclimate to temperature elevations, but enhanced their short-term acclimation capacity. Hence, we provide evidence that alternative splicing has a central role in the definition of plant fitness plasticity to stressful conditions.

    Stress response, peripheral serotonin and natural antibodies in feather pecking genotypes and phenotypes and their relation with coping style
    Eijk, Jerine A.J. van der; Lammers, Aart ; Kjaer, J.B. ; Rodenburg, T.B. - \ 2019
    Physiology and Behavior 199 (2019). - ISSN 0031-9384 - p. 1 - 10.
    Feather pecking - genotype - natural antibody - phenotype - serotonin - stress response

    Feather pecking (FP), a serious welfare and economic issue in the egg production industry, has been related to coping style. Proactive and reactive coping styles differ in, among others, the stress response, serotonergic activity and immune activity. Yet, it is unknown whether genetic lines divergently selected on FP (i.e. FP genotypes) or individuals differing in FP (i.e. FP phenotypes) can be categorized into coping styles. Therefore, we determined peripheral serotonin (5-HT) levels, natural antibody (NAb) titers, behavioral and corticosterone (CORT) responses to manual restraint (MR) in FP genotypes (high FP (HFP), low FP (LFP) and unselected control (CON) line) and FP phenotypes (feather pecker, feather pecker-victim, victim and neutral). We further examined the consistency of and relationships between behavioral and physiological measures. FP genotypes differed in behavioral responses to MR, 5-HT levels and NAb titers, but not in CORT levels after MR. HFP birds had less active responses at adolescent age, but more active responses at adult age compared to LFP and CON birds. The CON line had higher 5-HT levels at adolescent age, while the HFP line had lower 5-HT levels than the other lines at adult age. Overall, the HFP line had lower IgM NAb titers, while the LFP line had lower IgG NAb titers compared to the other lines. FP phenotypes differed in behavioral responses to MR and 5-HT levels, but not in CORT levels after MR or NAb titers. Within the HFP line, feather peckers tended to have less active responses compared to neutrals at adolescent age, while victims had more active responses compared to the other phenotypes at adult age. Feather peckers had higher 5-HT levels than neutrals at adult age. Behavioral and CORT responses to MR were not consistent over time, suggesting that responses to MR might not reflect coping style in this study. Furthermore, proactive behavioral responses were correlated with reactive physiological measures and vice versa. Thus, it was not possible to categorize FP genotypes or FP phenotypes into specific coping styles.

    Global DNA Compaction in Stationary-Phase Bacteria Does Not Affect Transcription
    Janissen, Richard ; Arens, Mathia M.A. ; Vtyurina, Natalia N. ; Rivai, Zaïda ; Sunday, Nicholas D. ; Eslami-Mossallam, Behrouz ; Gritsenko, Alexey A. ; Laan, Liedewij ; Ridder, Dick de; Artsimovitch, Irina ; Dekker, Nynke H. ; Abbondanzieri, Elio A. ; Meyer, Anne S. - \ 2018
    Cell 174 (2018)5. - ISSN 0092-8674 - p. 1188 - 1199.e14.
    DNA condensation - Dps - magnetic tweezers - nucleoid - RNA polymerase - single-molecule biophysics - stationary phase - stress response - transcription

    In stationary-phase Escherichia coli, Dps (DNA-binding protein from starved cells) is the most abundant protein component of the nucleoid. Dps compacts DNA into a dense complex and protects it from damage. Dps has also been proposed to act as a global regulator of transcription. Here, we directly examine the impact of Dps-induced compaction of DNA on the activity of RNA polymerase (RNAP). Strikingly, deleting the dps gene decompacted the nucleoid but did not significantly alter the transcriptome and only mildly altered the proteome during stationary phase. Complementary in vitro assays demonstrated that Dps blocks restriction endonucleases but not RNAP from binding DNA. Single-molecule assays demonstrated that Dps dynamically condenses DNA around elongating RNAP without impeding its progress. We conclude that Dps forms a dynamic structure that excludes some DNA-binding proteins yet allows RNAP free access to the buried genes, a behavior characteristic of phase-separated organelles. Despite markedly condensing the bacterial chromosome, the nucleoid-structuring protein Dps selectively allows access by RNA polymerase and transcription factors at normal rates while excluding other factors such as restriction endonucleases.

    Exploring the genetics underlying the responses to consecutive combinations of biotic stresses and drought in Arabidopsis thaliana
    Huang, Pingping - \ 2016
    Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - Wageningen : Wageningen University - ISBN 9789462578593 - 291
    arabidopsis thaliana - genetic models - stress - stress response - drought - botrytis - pieris (lepidoptera) - genetics - gene expression - arabidopsis thaliana - genetische modellen - stress - stressreactie - droogte - botrytis - pieris (lepidoptera) - genetica - genexpressie

    Plants growing in natural environments are exposed to a broad range of biotic (pathogen attack, insect herbivory, etc.) and abiotic factors (drought, extreme temperatures, UV radiation, salinity, etc.) that are known to cause stress symptoms in many species (Pareek et al., 2010; Robert-Seilaniantz et al., 2010). Biotic and abiotic stress-inducing determinants often adversely impact plant growth and development, frequently leading to severe annual yield losses in agricultural production (Pierik et al., 2013; Pieterse et al., 2012; Stam et al., 2014). In the research endeavors described in this thesis, Arabidopsis thaliana was used as a model organism to study plant responses to different sequential combinations of biotic factors (infection with Botrytis or herbivory by Pieris) and drought. The main objective was to identify genes that contribute to tolerance to the aforementioned sequential stress combinations. Genome-wide association (GWA) mapping and RNA sequencing (RNA-seq) approaches were used to identify combinatorial stress responsive genes. A number of candidate genes to combinatorial stress responses were identified by GWA analysis and RNA-seq. The physiological function of some candidate genes in different stress conditions were characterized using T-DNA insertion mutants and gene expression study. However, the physiological function of many allelic variants in stress conditions remain to be discovered. The study highlights the importance of an array of genes, crucial to the underlying defense processes, as targets for breeding by allele mining, ultimately aimed at improvement of crop tolerance to frequent combinations of stress factors.

    Supplementary data: Combined biotic and abiotic stress resistance in tomato
    Kissoudis, C. ; Chowdhury, Rawnaq ; Heusden, A.W. van; Wiel, C.C.M. van de; Finkers, H.J. ; Visser, R.G.F. ; Bai, Y. ; Linden, C.G. van der - \ 2016
    Wageningen UR
    solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
    Abiotic and biotic stress factors are the major constrains for the realization of crop yield potential. As climate change progresses, the spread and intensity of abiotic as well as biotic stressors is expected to increase, with increased probability of crops being exposed to both types of stress. Shielding crops from combinatorial stress requires a better understanding of the plant’s response and its genetic architecture. In this study, we evaluated resistance to salt stress, powdery mildew and to both stresses combined in tomato, using the S. habrochaites LYC4 introgression line (IL) population. The IL population segregated for both salt stress tolerance and powdery mildew resistance. Using SNP array marker data, QTLs were identified for salt tolerance as well as Na+ and Cl- accumulation. Salt stress increased the susceptibility of the population to powdery mildew in an additive manner. Phenotypic variation for disease resistance was reduced under combined stress as indicated by the coefficient of variation (CV). No correlation was found between disease resistance and Na+ and Cl- accumulation under combined stress Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance. These findings increase our understanding of the genetic regulation of responses to abiotic and biotic stress combinations and can provide leads to more efficiently breeding for tomatoes and other crops with a high level of disease resistance while maintaining their performance in combination with abiotic stress.
    Supplementary data: Responses to combined abiotic and biotic stress in tomato are governed by stress intensity and mechanism of resistance
    Kissoudis, C. ; Sri Sunarti, Sri ; Wiel, C.C.M. van de; Visser, R.G.F. ; Linden, C.G. van der; Bai, Y. - \ 2016
    Wageningen UR
    solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
    Stress conditions in agricultural ecosystems can occur in variable intensities. Different resistance mechanisms to abiotic stress and pathogens are deployed by plants. Thus, it is important to examine plant responses to stress combinations under different scenarios. Here, we evaluated the effect of different levels of salt stress ranging from mild to severe (50, 100 and 150mM NaCl) on powdery mildew (PM) resistance and overall performance of tomato introgression lines with contrasting levels of partial resistance, as well as isogenic lines carrying the PM resistance genes Ol-1 (associated with slow Hypersensitivity Response; HR), ol-2 (a mlo mutant associated with papilla formation) and Ol-4 (a R gene associated with fast HR). PM resistance was affected by salt stress in a genotype and stress intensity dependent manner. In susceptible and partial resistant lines, increased susceptibility was observed under mild salt stress (50mM) which was accompanied with accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced disease symptoms. Na+ and Cl- accumulation in the leaves was linearly related to the decreased pathogen growth under severe stress, suggesting a more direct role for the salt in suppressing PM growth. In contrast, complete resistance mediated by ol-2 and Ol-4 was unaffected under all treatment combinations, and was associated with a decreased growth penalty. Increased susceptibility and senescence under combined stress of the variety Moneymaker (MM) and the NIL Ol-1 was associated with the induction of ethylene and jasmonic acid pathway genes as well as of the cell wall invertase gene LIN6. These results highlight the significance of stress severity and resistance type on the plant’s performance under abiotic and biotic stress combination.
    Supplementary data: Hormone signalling regulation of tomato response to combined biotic and abiotic stress
    Kissoudis, C. ; Sri Sunarti, Sri ; Wiel, C.C.M. van de; Visser, R.G.F. ; Linden, C.G. van der; Bai, Y. - \ 2016
    Wageningen UR
    solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
    Plant hormones are paramount to plant adaptation to changing environmental conditions and interactions with microorganisms. There is currently limited knowledge on their significance in the response to stress combination. Using near isogenic lines (NILs) that carry the Ol-1, ol-2 and Ol-4 gene for resistance to tomato powdery mildew caused by Oidium neolycopersici, this study focused on the responses of these NILs to powdery mildew and salt stress combination. In these NILs, marker genes for monitoring hormonal pathways showed differential expression pattern upon powdery mildew infection. Further by crossing these NILs with tomato mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) the cross-talk among hormonal pathways was further investigated. Among the mutants, epinastic resulted in increased susceptibility of NIL-Ol-1 and breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition, effects that were more pronounced under combination with salt stress. On the other hand notabilis, resulting in H2O2 overproduction greatly reduced susceptibility of NIL-Ol-1 under combined stress accompanied however by heightened sensitivity to salt stress. Callose deposition reduction led to partial resistance breakdown in NIL-ol-2 which was reversed under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. We discuss the critical role that hormone signalling appears to have for the outcome of combined stress and powdery mildew in terms of resistance and plant fitness integrating observations from physiological, histochemical and gene expression analyses. These significant insights obtained extend our understanding of hormonal regulation of combined stress responses and can aid in narrowing down targets for improving crop performance under stress combinations.
    Supplementary data: Roles and contribution of tomato WRKY genes to salt stress and powdery mildew resistance
    Kissoudis, C. ; Gao, D. ; Pramanik, Dewi ; Birhanu, Mengistu ; Wiel, C.C.M. van de; Visser, R.G.F. ; Bai, Y. ; Linden, C.G. van der - \ 2016
    Wageningen UR
    solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysisplant breeding - salt tolerance
    WRKY is a transcription factor family unique to plants with diverse functions in defense pathways, abiotic stress tolerance and developmental programs. Family members are characterized by the conserved WRKY domain and significant sequence variation in the remainder of the protein, which is translated into distinct functions even for closely related genes. We utilized the extensive functional characterization of the Arabidopsis thaliana WRKY family to identify tomato homologues of Arabidopsis WRKY genes that are involved in defense responses (AtWRKY 11, 29, 48, 70 and 72). In total 13 tomato WRKY homologues were identified for these genes, of which 9 were successfully over-expressed, and 12 stably silenced via RNAi in transgenic tomato lines. The transgenic lines were evaluated for their response to salt stress, powdery mildew resistance and the combination of these stresses. Lines overexpressing SlWRKY11 and SlWRKY23, and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and improved salt tolerance. For SlWRKY11 and SlWRKY23 overexpression (OE) lines, this was accompanied by a moderate increase in oxidative stress tolerance. The SlWRKY6-OE line showed strongly improved salt stress tolerance, but a growth penalty under control conditions. Exceptional phenotypes were observed for the SlWRKY10-OE line (stunted growth) and the RNAi line SlWRKY23-RNAi (necrotic symptoms), but these phenotypes were partly restored to normal under salt stress. Both these lines exhibited increased resistance to powdery mildew, but this was compromised when the plants were put under salt-stress as well. Important functions for tomato WRKY genes were revealed in both the abiotic and biotic stress response and several genes should be further explored to elucidate their downstream regulatory functions that lead to increased stress tolerance.
    Ecogenomics of plant resistance to biotic and abiotic stresses
    Davila Olivas, N.H. - \ 2016
    Wageningen University. Promotor(en): Marcel Dicke; Joop van Loon. - Wageningen : Wageningen University - ISBN 9789462576575 - 259
    016-3932 - arabidopsis thaliana - defence mechanisms - drought resistance - insect pests - plant pathogenic fungi - stress - stress response - transcriptomics - genomics - genetic mapping - arabidopsis thaliana - verdedigingsmechanismen - droogteresistentie - insectenplagen - plantenziekteverwekkende schimmels - stress - stressreactie - transcriptomica - genomica - genetische kartering

    In natural and agricultural ecosystems, plants are exposed to a wide diversity of abiotic and biotic stresses such as drought, salinity, pathogens and insect herbivores. Under natural conditions, these stresses do not occur in isolation but commonly occur simultaneously. However, plants have developed sophisticated mechanisms to survive and reproduce under suboptimal conditions. Genetic screenings and molecular genetic assays have shed light on the molecular players that provide resistance to single biotic and abiotic stresses. Induced defenses are attacker specific and phytohormones play an essential role in tailoring these defense responses. Because phytohormones display antagonistic and synergistic interactions, the question emerges how plants elicit an effective defense response when exposed to conflicting signals under multiple attack. Recent studies have shed light on this issue by studying the effects of combinations of stresses at the phenotypic, transcriptomic and genetic level. These studies have concluded that the responses to combined stresses can often not be predicted based on information about responses to the single stress situations or the phytohormones involved. Thus, combined stresses are starting to be regarded as a different state of stress in the plant. Studying the effects of combinations of stresses is relevant since they are more representative of the type of stresses experienced by plants in natural conditions.

    In a coordinated effort, responses of Arabidopsis thaliana to a range of abiotic and biotic stresses and stress combinations have been explored at the genetic, phenotypic, and transcriptional level. For this purpose we used an ecogenomic approach in which we integrated the assessment of phenotypic variation and Genome-Wide Association (GWA) analysis for a large number of A. thaliana accessions with an in-depth transcriptional analysis. The focus of this thesis is especially on (but not limited to) three stresses, i.e. drought, herbivory by Pieris rapae caterpillars, and infection by the necrotrophic fungal pathogen Botrytis cinerea. These stresses were chosen because the responses of A. thaliana to these three stresses are highly divergent but at the same time regulated by the plant hormones JA and/or ABA. Consequently, analysis of responses to combinatorial stresses is likely to yield information on signaling nodes that are involved in tailoring the plant’s adaptive response to combinations of these stresses. Responses of A. thaliana to other biotic and abiotic stresses are included in an integrative study (Chapter 6).

    We first investigated (Chapter 2) the extent of natural variation in the response to one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips, Myzus persicae aphids) and two combined stresses (drought plus P. rapae, and B. cinerea plus P. rapae). Using 308 A. thaliana accessions originating from Europe, the native range of the species, we focused on the eco-evolutionary context of stress responses. We analyzed how the response to stress is influenced by geographical origin, genetic relatedness and life-cycle strategy, i.e. summer versus winter annual. We identified heritable genetic variation for responses to the different stresses. We found that winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars and to the combined stresses of drought followed by P. rapae and infection by the fungus B. cinerea followed by herbivory by P. rapae. Furthermore, we found differential responses to drought along a longitudinal gradient.

    We further investigated, using A. thaliana accession Col-0, how phenotypic and whole-genome transcriptional responses to one stress are altered by a preceding or co-occurring stress (Chapters 3 and 4). The whole-transcriptomic profile of A. thaliana triggered by single and combined abiotic (drought) and biotic (herbivory by caterpillars of P. rapae, infection by B. cinerea) stresses was analyzed by RNA sequencing (RNA-seq). Comparative analysis of plant gene expression triggered by single and double stresses revealed a complex transcriptional reprogramming. Mathematical modelling of transcriptomic data, in combination with Gene Ontology analysis highlighted biological processes specifically affected by single and double stresses (Chapters 3). For example, ethylene (ET) biosynthetic genes were induced at 12 h by B. cinerea alone or drought followed by B. cinerea inoculation. This induction was delayed when plants were pretreated with P. rapae by inducing ET biosynthetic genes only 18 hours post inoculation. Other processes affected by combined stresses include wound response, systemic acquired resistance (SAR), water deprivation and ABA response, and camalexin biosynthesis.

    In Chapter 4, we focused on the stress imposed by P. rapae herbivory alone or in combination with prior exposure to drought or infection with B. cinerea. We found that pre-exposure to drought stress or B. cinerea infection resulted in a significantly different timing of the caterpillar-induced transcriptional changes. Additionally, the combination of drought and P. rapae induced an extensive downregulation of A. thaliana genes involved in defence against pathogens. Despite the larger reduction in plant biomass observed for plants exposed to drought plus P. rapae feeding compared to P. rapae feeding alone, this did not affect weight gain of this specialist caterpillar.

    In Chapter 5, we used univariate GWA to (1) understand the genetic architecture of resistance to the different stresses and (2) identify regions of the genome and possible candidate genes associated with variation in resistance to those stresses. In Chapter 5 a subset of the stresses addressed in Chapter 1 (i.e. drought, herbivory by P. rapae and P. xylostella, and the combined stresses drought plus P. rapae and B. cinerea plus P. rapae) were investigated. Results from GWA were integrated with expression data generated in Chapters 3 and 4 or available from the literature. We identified differences in genetic architecture and QTLs underlying variation in resistance to (1) P. rapae andP. xylostella and (2) resistance to P. rapae and combined stresses drought plus P. rapae and B. cinerea plus P. rapae. Furthermore, several of the QTLs identified contained genes that were differentially expressed in response to the relevant stress. For example, for P. xylostella one of the QTLs contained only two genes encoding cysteine proteases (CP1 and CP2). The expression data indicated that these genes were induced by P. rapae and P. xylostella herbivory.

    In Chapter 6, the genetic architecture underlying plant resistance to 11 single stresses and some of their combinations was investigated. First, the genetic commonality underlying responses to different stresses was investigated by means of genetic correlations,, revealing that stresses that share phytohormonal signaling pathways also share part of their genetic architecture. For instance, a strong negative genetic correlation was observed between SA and JA inducers. Furthermore, multi-trait GWA identified candidate genes influencing the response to more than one stress. For example, a functional RMG1 gene seems to be associated with susceptibility to herbivory by P. rapae and osmotic stress since loss of function mutants in RMG1 displayed higher resistance to both stresses. Finally, multi-trait GWA was used to identify QTLs with contrasting and with similar effects on the response to (a) biotic or abiotic stresses and (b) belowground or aboveground stresses.

    Finally, In Chapter 7, I discuss the feasibility of obtaining plants that are resistant to multiple stresses from the point of view of genetic trade-offs and experimental limitations. The ecogenomic approach for gene discovery taken in this thesis is discussed, and recommendations are especially given on the use of herbivorous insects in quantitative genetic studies of stress resistance. Furthermore, alternatives to the use of insects in quantitative genetic studies of stress resistance are discussed and proposed. Finally, I discuss the feasibility of using an ecogenomic approach to study stress responses in other plant species than the model plant of molecular genetics, A. thaliana.

    A wealth of candidate genes was generated by taking an ecogenomic approach, in particular transcriptome analysis and GWA analysis. Functional characterization of these genes is a next challenge, especially in the context of multiple stress situations. These genes constitute a rich source of potential factors important for resistance to abiotic, biotic and combined stresses that in the future may be applied for crop improvement.

    Genetics and regulation of combined abiotic and biotic stress tolerance in tomato
    Kissoudis, C. - \ 2016
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Gerard van der Linden; Yuling Bai. - Wageningen : Wageningen University - ISBN 9789462576568 - 212
    solanum lycopersicum - tomatoes - disease resistance - stress tolerance - defence mechanisms - plant diseases - abiotic injuries - stress response - phenotypic variation - genetic analysis - plant breeding - salt tolerance - solanum lycopersicum - tomaten - ziekteresistentie - stresstolerantie - verdedigingsmechanismen - plantenziekten - abiotische beschadigingen - stressreactie - fenotypische variatie - genetische analyse - plantenveredeling - zouttolerantie

    Projections on the impact of climate change on agricultural productivity foresee prolonged and/or increased stress intensities and enlargement of a significant number of pathogens habitats. This significantly raises the occurrence probability of (new) abiotic and biotic stress combinations. With stress tolerance research being mostly focused on responses to individual stresses, our understanding of plants’ ability to adapt to combined stresses is limited.

    In an attempt to bridge this knowledge gap, we hierarchized in chapter 1 existing information on individual abiotic or biotic stress adaptation mechanisms taking into consideration different anatomical, physiological and molecular layers of plant stress tolerance and defense. We identified potentially crucial regulatory intersections between abiotic and biotic stress signalling pathways following the pathogenesis timeline, and emphasized the importance of subcellular to whole plant level interactions by successfully dissecting the phenotypic response to combined stress. We considered both explicit and shared adaptive responses to abiotic and biotic stress, which included amongst others R-gene and systemic acquired resistance as well as reactive oxygen species (ROS), redox and hormone signalling, and proposed breeding targets and strategies.

    In chapter 3 we focused on salt stress and powdery mildew combination in tomato, a vegetable crop with a wealth of genetic resources, and started with a genetic study. S. habrochaites LYC4 was found to exhibit resistance to both salt stress and powdery mildew. A LYC4 introgression line (IL) population segregated for both salt stress tolerance and powdery mildew resistance. Introgressions contributing to salt tolerance, including Na+ and Cl- accumulation, and powdery mildew resistance were precisely pinpointed with the aid of SNP marker genotyping. Salt stress (100mM NaCl) combined with powdery mildew infection increased the susceptibility of the population to powdery mildew in an additive manner, while decreasing the phenotypic variation for this trait. Only a few overlapping QTLs for disease resistance and salt stress tolerance were identified (one on a short region at the top of Chromosome 9 where numerous receptor-like kinases reside). Most genetic loci were specific for either salt stress tolerance or powdery mildew resistance indicating distinct genetic architectures. This enables genetic pyramiding approaches to build up combined stress tolerance.

    Considering that abiotic stress in nature can be of variable intensities, we evaluated selected ILs under combined stress with salinity ranging from mild to severe (50, 100 and 150mM NaCl) in chapter 4. Mild salt stress (50mM) increased powdery mildew susceptibility and was accompanied by accelerated cell death-like senescence. On the contrary, severe salt stress (150mM) reduced the disease symptoms and this correlated with leaf Na+ and Cl- content in the leaves. The effects of salt stress on powdery mildew resistance may be dependent on resistance type and mechanisms. Near Isogenic Lines (NILs) that carry different PM resistance genes (Ol-1 (associated with slow hypersensitivity response, HR), ol-2 (an mlo mutant associated with papilla formation) and Ol-4 (an R gene associated with fast HR) indeed exhibited differential responses to combined stress. NIL-Ol-1 resembled the LYC4 ILs response, while NIL-ol-2 and NIL-Ol-4 maintained robust resistance and exhibited no senescence symptoms across all combinations, despite the observed reduction in callose deposition in NIL-ol-2. Increased susceptibility, senescence and fitness cost of NIL-Ol-1 under combined stress coincided with high induction of ethylene and jasmonate biosynthesis and response pathways, highly induced expression of cell wall invertase LsLIN6, and a reduction in the expression of genes encoding for antioxidant enzymes. These observations underlined the significance of stress intensity and mechanism of resistance to the outcome of salt stress and powdery mildew combination, underscoring the involvement of ethylene signalling to the susceptibility response under combined stress.

    To examine the significance of hormone signalling in combined stress responses we evaluated crosses of tomato hormone mutants notabilis (ABA-deficient), defenseless1 (JA-deficient) and epinastic (ET overproducer) with NIL-Ol-1, NIL-ol-2 and NIL-Ol-4 in chapter 5. The highly pleiotropic epinastic mutant increased susceptibility of NIL-Ol-1, but decreased the senescence response under combined stress, and resulted in partial breakdown of NIL-ol-2 resistance, accompanied by reduced callose deposition. The effects of ET overproduction on susceptibility were more pronounced under combined stress. ABA deficiency in notabilis on the other hand greatly reduced susceptibility of NIL-Ol-1under combined stress at the expense of stronger growth reduction, and induced ROS overproduction. Partial resistance breakdown in the ol-2xnotabilis mutant accompanied by reduced callose deposition was observed, and this was restored under combined stress. Jasmonic acid deficiency phenotypic effects in defenseless mutants were subtle with modest increase in susceptibility for NIL-Ol-1 and NIL-ol-2. For NIL-ol-2 this increased susceptibility was reverted under combined stress. NIL-Ol-4 resistance remained robust across all mutant and treatment combinations. These results highlight the catalytic role of ET and ABA signalling on susceptibility and senescence under combined stress, accentuating concomitantly the importance of signalling fine tuning to minimize pleiotropic effects.

    The potential of exploiting transcription factors to enhance tolerance to multiple stress factors and their combination was investigated in chapter 6 through the identification and functional characterization of tomato homologues of AtWRKYs 11, 29, 48, 70 and 72. Thirteen tomato WRKY homologues were identified, of which 9 were overexpressed (using transformation with A. tumefaciens) and 12 stably silenced via RNAi in tomato cultivar Money Maker (MM). SlWRKY11-OE and SlWRKY23-OE overexpressors and RNAi lines of SlWRKY7 and SlWRKY9 showed both increased biomass and relative salt tolerance. SlWRKY6-OE exhibited the highest relative salt stress tolerance, but had strongly decreased growth under control conditions. Exceptional phenotypes under control conditions were observed for SlWRKY10-OE (stunted growth) and SlWRKY23-RNAi (necrotic symptoms). These phenotypes were significantly restored under salt stress, and accompanied by decreased ROS production. Both lines exhibited increased resistance to powdery mildew, but this resistance was compromised under salt stress combination, indicating that these genes have important functions at the intersection of abiotic and biotic stress adaptation. SlWRKY23 appears to have a key regulatory role in the control of abiotic stress/defense and cell death control.

    Experimental observations are critically discussed in the General Discussion with emphasis on potential distinctive responses in different pathosystems and abiotic and biotic stress resistance mechanisms as well as genetic manipulations for effectively achieving combined stress tolerance. This includes deployment of individual common regulators as well as pyramiding of non-(negatively) interacting components such as R-genes with abiotic stress resistance genes, and their translation potential for other abiotic and biotic stress combinations. Understanding and improving plant tolerance to stress combinations can greatly contribute to accelerating crop improvement towards sustained or even increased productivity under stress.

    Endogene stress : stressoren (deel 3)
    Leenstra, S.H. ; Scheerboom, J. - \ 2014
    Aquacultuur 29 (2014)4. - ISSN 1382-2764 - p. 22 - 23.
    aquacultuur - vissen - stressreactie - stress omstandigheden - aquaculture - fishes - stress response - stress conditions
    Een vishouder dient te weten dat een vis gedurende het leven ontwikkelingen doormaakt, die stress voor het dier betekenen. Hierbij worden - ter afsluiting van vorige bijdragen over stressoren (zie Aquacultuur 2014, nr. 2 en 3) - voorbeelden van endogene stress beschreven.
    Over ongekende gevoeligheden van vissen : vissen en hun stressoren (deel 2)
    Leenstra, S.H. ; Scheerboom, J. - \ 2014
    Aquacultuur 29 (2014)3. - ISSN 1382-2764 - p. 6 - 10.
    vissen - stressfactoren - stressreactie - natuurlijke toxinen - formaldehyde - waterkwaliteit - aquacultuur - fishes - stress factors - stress response - natural toxins - formaldehyde - water quality - aquaculture
    Zoals bekend, streeft een viskweker naar continue groei, optimale voederconversie en een geplande productietijd. Stress verlaagt bij vissen de immunologische weerstand, waardoor ziektes kunnen uitbreken en een lagere productie het gevolg is. Daarom worden stressoren op een kwekerij zo veel mogelijk gemeden. Hiertoe is het allereerst nodig dat een viskweker weet welke stressoren zich op de kwekerij kunnen voordoen en vervolgens hoe gevoelig de vis hiervoor is.
    The fearful feather pecker : applying the principles to practice to prevent feather pecking in laying hens
    Haas, E.N. de - \ 2014
    Wageningen University. Promotor(en): Bas Kemp; A.G.G. Groothuis, co-promotor(en): Bas Rodenburg; Liesbeth Bolhuis. - Wageningen : Wageningen University - ISBN 9789462570429 - 285
    hennen - verenpikken - bangheid - gedragsproblemen - lijnen - hormonale controle - stressreactie - ontogenie - legresultaten - dierenwelzijn - diergedrag - dierfysiologie - hens - feather pecking - fearfulness - behaviour problems - lines - hormonal control - stress response - ontogeny - laying performance - animal welfare - animal behaviour - animal physiology

    Billions of laying hens are kept worldwide. Severe feather pecking (SFP) is a behaviour which occurs with a high prevalence on commercial farms. SFP, the pecking and plucking of feathers of another bird, induces pain and stress and can ultimately lead to cannibalism. Moreover, SFP can occur if a bird is unable to cope with fear and stress and is living in an inappropriate environment. SFP thus reduces the welfare of many laying hens worldwide. To prevent SFP it is essential to know the risk factors in its development. To that aim, first, two experimental studies were conducted to gain insight in the principles of SFP, and three on-farm studies were conducted to assess the risk factors of SFP under commercial conditions.


    Factors which relate to SFP are high fearfulness as young and low levels of brain and peripheral serotonin (5-HT) and brain dopamine (DA). Furthermore, commercial laying hen lines can differ in SFP tendencies and associated traits indicating that SFP has a genetic component. In chapters 2 and 3, fear response as young and adult, and stress response, 5-HT and DA brain levels as adult were

    compared in hens of two lines: the low mortality line (LML) selected on low levels of mortality due to cannibalism and individual performance vs. the control line (CL) which was selected on individual performance only. Hens were exposed to an Open Field (OF) test at 5 weeks of age and a Manual Restraint (MR) test at 33 weeks of age. At 33 weeks of age, levels of corticosterone (CORT) post MR and 5-HT and DA levels in four brain areas were determined. Hens of the LML were less fearful at both ages and had lower levels of DA in the arcopallium, a somatomotor area involved in fear and motor control, compared to hens of the CL. In chapter 2, it was also shown that fearful chicks had higher levels of CORT and higher activity levels as adult, compared to non-fearful chicks. Moreover,

    presence of fearful animals in the group was related to average CORT levels of their pen members. Fearful hens may induce social instability in a group, and thereby affecting the stress-sensitivity of their group mates. These results indicate that groups differ in levels of fear and stress-sensitivity, and that fearfulness at a young age can lead to stress-sensitivity as adults, which create a risk for development of SFP.


    In chapters 4, 5 and 6, the laying hen production chain consisting of parent stock, rearing flocks and laying flocks was studied. Risk factors for SFP could originate from previous parts in the chain. Therefore, in all on-farm studies, measurements of SFP, fearfulness, basal CORT and peripheral 5-HT system were obtained, and related to housing conditions and to previous parts in the chain. Fearfulness was assessed, on a flock level, by distance to a stationary person (SP) test and latency

    of bird to approach a novel object (NO). Dekalb White (DW) and ISA brown (ISA) crosses whose pure lines differ in levels of fear, CORT, 5-HT and DA, were compared. First, parent stock (PS) flocks were studied and associations between production performance and measurements of fear, stress and 5-HT were conducted and related to group size conditions (chapter 4). Second, rearing flocks originating from PS flocks were studied throughout the rearing period (chapter 5). High levels of feather damage, CORT and 5-HT in the mothers were related to fearfulness and SFP in their offspring at flock level. Especially, a large flock size and limitation and/or disruption in litter supply affected SFP and levels of fearfulness and 5-HT (chapter 5). Finally, high levels of feather damage during the laying period were related to high SFP rearing, and high fearfulness during rearing and laying (chapter 6). These studies together aimed to determine the risk factors for the development of SFP and the resulting feather damage. The main outcomes of these studies are as follows.

    Ø Parent stock flocks

    DW flocks were more fearful of an SP and hens had higher levels of feather damage than in ISA flocks. ISA flocks, in turn, were more fearful of the NO and hens had higher 5-HT levels than in DW flocks. A small flock size led to higher feed conversion, mortality levels, and smothering events in ISA but not in DW flocks. These results indicate that DW and ISA PS flocks differ in levels of fear and

    feather damage, and respond differently to their social environment. For both crosses, fear of an SP related to high mortality and fear of the NO related to low hen body weight, egg weight, and feed intake. High basal CORT related to low egg weight. High fear and stress levels in PS flocks may, thus, negatively affect (re)production, and thereby potentially negatively influence the developing


    Ø Rearing flocks

    In the DW cross, high CORT, feather damage, and 5-HT of mother hens related to high SFP and fearfulness of their rearing flocks at 1 week of age. At 5 weeks of age, a peak in both gentle feather pecking (GFP) and SFP was recorded, coinciding with a disruption in substrate availability (i.e. a temporal absence of substrate) and a limitation of substrate (i.e. limited amounts of substrate

    provided) in some of the farms. Especially, ISA pullets showed higher SFP under substrate limitation and became more fearful under substrate disruption than DW pullets. ISA pullets had higher 5-HT levels than DW pullets. Only in the ISA cross, high 5-HT related to high fearfulness, specifically under substrate disruption. For both crosses, high fearfulness was related to high feather damage. Furthermore, in a level system (floor system where levels are gradually added) higher levels of SFP and feather damage were found compared to an aviary system (a tier-system with cages and litter area). These results highlight that; 1) parental effects exist in the development of fearfulness and SFP, 2) disruption and limitation in substrate availability can lead to high SFP at 5 weeks of age, 3) ISA pullets are more strongly influenced by environmental conditions than DW pullets and 4) a level housing, which coincided with a large group size, increase the risk of SFP and feather damage during rearing.

    Ø Laying flocks

    In our sample, 49% of the laying flocks had severe damage at 40 weeks of age, compared with 71%, 65% and 53% of the rearing flocks at 15, 10 and 5 weeks of age, respectively. High fear of a SP at rearing and high SFP at 5 weeks of age related to high levels of feather damage at lay. In a floor system and at a large flock size higher levels of feather damage were recorded than in an aviary system and at a small flock size. An adjusted management on the laying farm (i.e. aerated blocks, presence of roosters or a radio playing) reduced levels of feather damage compared to standard management. DW flocks were more fearful of the SP and NO than ISA flocks. This study showed that factors during rearing and laying contributed to feather damage at 40 weeks of age.

    With the knowledge from the experimental and on-farm studies in this thesis, an assessment of the risk factors for SFP could be established. Risk factors for SFP are: high fear, stress and feather damage in DW parent stock, high fear of humans, especially for DW hens, litter disruption or limitation during rearing, large group sizes, and a floor or level system.

    Worms under stress: unravelling genetic complex traits through perturbation
    Rodriguez Sanchez, M. - \ 2014
    Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Jan Kammenga. - Wageningen : Wageningen University - ISBN 9789461738516 - 124
    caenorhabditis elegans - genetica - diermodellen - stress - warmtestress - stressreactie - genetische variatie - caenorhabditis elegans - genetics - animal models - stress - heat stress - stress response - genetic variation

    The genetic architecture of an organism could be considered ‘the most amazing piece of engineering’ existing in nature. Looking from a certain distance, the genetic complexity of an organism could be described as an immense jigsaw puzzle. As in a real jigsaw, the connection between two pieces will suggest or bring us to the third one and so on. The genetic responses to perturbation reveal interactions between many alleles of which effects are not that noticeable under optimal conditions.

    In the general introduction of this thesis (chapter 1), the reader can find a brief overview of the model organismCaenorhabditis elegans and how this nematode has increasingly taken a crucial position in relevant biological studies over the last few decades. Moreover, the outline of this thesis is extensively described in this section.

    My thesis brings together examples of perturbation in C. elegans at three different levels: phenotypic perturbation, as a result of genetic variation in Recombinant Inbred Lines (RILs), genetic perturbation, performed by genome combinations (RILs), introgressed fragments of exogenous genome in introgression lines (ILs) and induced mutations, and environmental perturbation (i.e. heat stress conditions). We have combined these perturbations in order to increase the potential of these tools in investigating genetic variation in C. elegans. Our RILs and ILs are combinations of two of the most divergent genomes of C. elegans strains these are Bristol N2 and CB4856 (Hawaii).

    The translation of this knowledge to research in human disease is of great interest since many complex diseases are regulated by small effect genes that occur during stress or aging. This thesis expounds how C. elegans has gained a prominent position as a model organism for studying the genetic of complex disease pathways. Here I have presented different studies and concluded that environmental stress perturbation experiments in C. elegans represents a crucial implement in the way to increase our understanding of the genetics of human diseases such as Huntington’s disease, Parkinson’s disease, Alzheimer’s disease or cancer. We performed an exhaustive analysis of the current experiments and new insights that used C. elegans as a model organism for complex human diseases. This thesis contains a detailed study where we analyze the past, discuss about the present and suggest new possibilities for the future of C. elegans as a model organism (chapter 2).

    The experimental sections of the thesis consist of quantitative genetic analyses of genomic regions associated with phenotypic variations observed under a certain perturbation. We use genotypic and phenotypic data measured in our RILs and ILs together with QTL mapping techniques to estimate the position of candidate loci, which are associated with a phenotypic variation.

    In C. elegans, β-catenin genebar-1plays an important role in vulva development whereas in humans it plays an important role in cancer progression.In our study in bar-1 mutant RILs, we constructed different combinations of two genomes in C. elegans strains. The lack of β-catenin was included to the combination of the two genomes. These mutant RILs point us to the presence of polymorphic modifiers of vulva development in C. elegans and pave the way towards the development of a new tool to uncover cryptic variation in two genetic variants (chapter 3).

    To gain insight into the subtle effects of natural variants and more concretely into the genetics of heat-shock recovery, we exposed RILs to heat-stress. We investigated here the hormesis effects on life span and offspring, and described associated loci in C. elegans (chapter 4). Our observations showed that there is natural variation in hormesis effects on life span for heat-shock.

    As a combination of environmental and genetic perturbation, we studied the effects of heat stress in C. elegans combined with ILs (chapter 5). We demonstrate the importance of the chromosome IV and a concrete locus in heat stress response. In order to identify the role of a locus distal to chromosomeIV, we selected a set of ILs containing a fragment of CB4856 (Hawaii) genome introgressed in a N2 (Bristol) background around this candidate locus previously described in other studies. We studied gene expression in normal and heat stress conditions by quantitative RT-PCR. Through this study we are able to confirm the role of the locus distal to chromosome IV in heat stress response.

    In the general discussion (chapter 6) I discuss the final results of the thesis and put them into the perspective of the current progresses in genetics studies of C. elegans and how stress response is associated with variations in pathways and thus in cellular and molecular process in health and/or disease.

    Overall this thesis demonstrates at least a part of the incredible potential of C. elegans as a complex genetic model using quantitative genetic analyses, which complement more established forward genetic screens.

    Genetic analysis of drought stress response in Arabidopsis thaliana and Brassica rapa
    El-Soda, M. - \ 2013
    Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Mark Aarts. - S.l. : s.n. - ISBN 9789461737441 - 146
    brassica campestris - arabidopsis thaliana - droogte - droogteresistentie - stressreactie - genetische analyse - genotypen - genotype-milieu interactie - brassica campestris - arabidopsis thaliana - drought - drought resistance - stress response - genetic analysis - genotypes - genotype environment interaction

    Drought is the major abiotic stress affecting plant growth and limiting crop productivity worldwide. Plants have evolved three adaptive strategies, drought escape, drought avoidance and drought tolerance, to cope with drought. Knowledge on how Quantitative Trait Loci (QTL), or genes underlying these strategies interact with their environments will significantly increase our understanding and the success of breeding for drought tolerance. This thesis focused on phenotyping shoot and root traits ofA. thaliana and B. rapa grown on sand and in greenhouses,to further understand how plants can adapt to natural drought stress. In chapter 2, an already existing ArabidopsisRIL population was selected based on the differential root drought response of the two parental lines, Sha and Col, to be evaluated under different water regimes. Chapter 3 illustrated the use of GWAS in identifying candidate genes that are associated with pant response to drought.. In order to apply the same methodology in crop breeding, chapter 4 introduces a contribution to the genetic mapping of a new B. rapa RIL population, consisting of 160 lines and genotyped with 270 different markers was achieved. The morphological and physiological responses of this population to drought was evaluated in chapter 5. The results presented in the present thesis demonstrate that QxE is an important component of the genetic variance and can play a great role in improving drought tolerance in future breeding programs. In general, several QTL and SNPs were mapped either with main effect or with interaction with environments QxE. Many of the mapped QTL showed conditional neutrality and antagonestic pleiotropy.

    Stress responses and digestive tract robustness of Lactobacillus plantarum
    Bokhorst-van de Veen, H. van - \ 2013
    Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): P.A. Bron. - [S.l. : S.n. - ISBN 9789461736291 - 192
    lactobacillus plantarum - stressreactie - spijsverteringskanaal - genregulatie - adaptatie - lactobacillus plantarum - stress response - digestive tract - gene regulation - adaptation

    Lactobacillus plantarumis one of the most versatile lactic acid bacteria that can successfully inhabit a variety of environmental niches. It is a common inhabitant of the human and animal gastrointestinal (GI) tract and it is used as starter culture in various fermentation processes for different food raw-materials, including milk, fruits, vegetables, and meat. Moreover, L. plantarum is marketed as a health-promoting culture, i.e. a probiotic. In these different environments and processes the bacteria encounter stress conditions, such as heat, cold, acid, salt, and oxygen stress. Since starter cultures and probiotics require metabolic activity to contribute to the taste and texture of the fermented products, and/or viability to exert their in situ beneficial effect on the consumer, it is important to understand and improve the gene-regulatory adaptation that sustains their function and viability under these challenging conditions. Nowadays, genomic approaches are available that enable the global, genome-wide analysis of stress responses in lactic acid bacteria. The work presented in this thesis employs such tools and also developed some novel strategies to understand stress responses in L. plantarum.

    During wine fermentation, L. plantarum is exposed to ethanol and global transcriptome profiling demonstrated the gene expression adaptation of this microorganism upon short and long term exposure to sublethal levels of this solvent. The results suggested that the ethanol induced activation of the CtsR-related stress regulon contributes to its adaptation to ethanol exposure which also provides cross-protection against heat stress. Transcriptome analyses under different growth conditions of gene deletion derivatives of the L. plantarum WCFS1 strain that lack the genes encoding the stress response regulators ctsR and/or hrcA, enabled the refinement of the gene regulation repertoire that is controlled by these central regulators of stress responses in this species. Notably, the deletion of both stress-regulators, elicited transcriptome changes that affected a large variety of additional gene-functions in a temperature-dependent manner, which prominently included genes related to cell-envelope remodelling.

    Culturing of L. plantarum WCFS1 under different fermentation conditions led to large differences in GI-tract survival and robustness, which was addressed using a simple in vitro survival assay. Enhanced GI-tract survival and robustness could be associated with low salt and low pH conditions during the fermentations. The transcriptomes obtained for each of the fermentation conditions employed, were correlated with the observed GI-tract survival rates, enabling the identification of candidate genes involved in the robustness phenotype, including a transcription regulator involved in capsular polysaccharide remodelling (Lp_1669), a penicillin-binding protein (Pbp2A) involved in peptidoglycan biosynthesis, and a Na+/H+ antiporter (NapA3). A role of these candidate genes in actual survival in the GI-tract assay could be confirmed by mutation analysis, further confirming their contribution to GI-tract stress robustness in L. plantarum.

    This thesis also describes the use of a novel, next-generation sequencing-based method, for the assessment of the in vivo GI-tract persistence of different L. plantarum strains that were administered to healthy human volunteers in specifically designed strain-mixtures. A remarkable consistency of the strain-specific in vivo persistence curves was observed when comparing data obtained from different volunteers. Moreover, a striking congruency was observed between the strain-specific in vivo persistence curves and the predicted GI-tract survival based on the simple in vitro assay. Finally, evolutionary adaptation of L. plantarum WCFS1 to the murine GI-tract was studied by extended exposure of the strain to the mice digestive tract through consecutive rounds of (re)feeding of the longest persisting bacterial colonies. Re-sequencing of the genomes of more persistent derivatives of the original strain, and the evaluation of the genomic modifications identified, implied that genes encoding cell envelope-associated functions and energy metabolism play an important role in the determination of GI-tract persistence in L. plantarum.

    The results described in this thesis strive to obtain an improved understanding of the gene-regulatory adaptations of L. plantarum that allow its survival under stress conditions, including those associated with residence in the gastrointestinal tract of animals and humans, with the intention to exploit such understanding to rationally improve the robustness of these bacteria.

    Quantification of Bacillus cereus stress responses
    Besten, H.M.W. den - \ 2010
    Wageningen University. Promotor(en): Marcel Zwietering; Tjakko Abee, co-promotor(en): Roy Moezelaar. - [S.l. : S.n. - ISBN 9789085857143 - 216
    bacillus cereus - stressreactie - zoutgehalte - zouttolerantie - hittetolerantie - adaptatie - weerstand - stresstolerantie - bacillus cereus - stress response - salinity - salt tolerance - heat tolerance - adaptation - resistance - stress tolerance
    The microbial stability and safety of minimally processed foods is controlled by a deliberate combination of preservation hurdles. However, this preservation strategy is challenged by the ability of spoilage bacteria and food-borne pathogens to adapt to stressing environments providing cell robustness. Bacillus cereus is a toxin-producing, spore-forming bacterium, and is able to survive minimal processing conditions. A quantitative approach was followed to gain insight in B. cereus’ stress adaptive behavior at population, individual cell and molecular level.
    B. cereus’ ability to adapt to salt stress and gain robustness towards subsequent heat challenge-stress exposure was quantified in detail using primary kinetics models. The adaptive salt stress response was influenced by the adaptation-stress concentration, the growth phase of the cells, strain diversity and the culturing temperature during adaptation-stress treatment. The nonlinear nature of the heat inactivation kinetics suggested heterogeneity within the population with respect to stress adaptive behavior. The direct-imaging-based Anopore technology was used to quantitatively describe the population heterogeneity of B. cereus upon mild and severe salt stress treatments and during low temperature growth. Fluorescent labeling of cells provided insights in the origin of stress-induced population heterogeneity. Then, to elucidate adaptive salt stress responses at molecular level, the genome-wide transcriptome profiles of mildly and severely salt-stressed cells were compared. Various transcriptome responses could be correlated to phenotypic features of salt stress-adapted cells. Comparison of the transcriptome profiles of salt stress-adapted cells to those that were exposed to mild heat, acid and oxidative stress, directed to potential cellular biomarkers for stress adaptation. The selected candidate-biomarkers  the transcriptional regulator B (activating general stress responses), catalases (removing reactive oxygen species), and chaperones and proteases (maintaining protein quality)  were measured upon adaptation-stress treatment at transcript, protein and/or activity level, and their induction was correlated to adaptation-stress induced robustness towards challenge-stress. Various candidate-biomarkers were suitable to predict the robustness level of adaptation-stress pretreated cells towards challenge-stress, and are therefore potential predictive cellular indicators for adaptation-stress induced robustness. The predictive potential of transcripts differed from that of proteins and activity level, underlining the significance to evaluate predictive potential of candidate-biomarkers at different functional cell levels. This quantitative understanding of B. cereus’ stress adaptive behavior provides mechanistic insights and opens up avenues to come to a mechanism-based approach for designing mild preservation strategies.
    Use of genomic information in mass-spawning fish
    Massault, C. - \ 2010
    Wageningen University. Promotor(en): Johan van Arendonk, co-promotor(en): Henk Bovenhuis; D.J. de Koning. - [S.l. : S.n. - ISBN 9789085856887 - 172
    loci voor kwantitatief kenmerk - aquacultuur - dicentrarchus labrax - pagrus aurata - kuitschieten - morfometrie - stressreactie - pasteurellose - selectief fokken - genexpressieanalyse - diergenetica - quantitative trait loci - aquaculture - dicentrarchus labrax - pagrus aurata - spawning - morphometrics - stress response - pasteurellosis - selective breeding - genomics - animal genetics
    This thesis uses the current genetic and genomic resources to permit genetic improvement of stress response in European sea bass and disease resistance in gilthead sea bream, two traits of economical importance. One way to integrate such genetic knowledge into breeding programmes, is to detect for quantitative trait loci (QTLs), which are regions of the genome influencing a specific trait. The first step was to design successful experiments to detect QTLs in aquaculture species. The next part of the thesis focuses on QTL mapping in natural mating mass-spawning species, which have complex population structure (variable family sizes). Finally, the possibilities of using genomic selection for mass-spawning species, where natural mating is used, were investigated.
    Bacillus cereus acid stress responses
    Mols, J.M. - \ 2009
    Wageningen University. Promotor(en): Tjakko Abee; Marcel Zwietering, co-promotor(en): Roy Moezelaar. - [S.l. : S.n. - ISBN 9789085854944 - 176
    bacillus cereus - stressreactie - voedselbewaring - bacillus cereus - stress response - food preservation
    Bacillus cereus is a ubiquitous Gram-positive organism, which frequently causes foodborne
    illnesses. The widespread prevalence of B. cereus makes it a common contaminant in
    fresh foods where it also can cause spoilage. To prevent food-borne diseases and food
    spoilage, foods are often processed and/or preserved. In recent years, consumers’
    preferences have directed to fresher and tastier foods and this has acted as a driver for food
    industry to use milder processing and preservation techniques. Examples of hurdles that can
    be applied to preserve foods are low pH and the addition of organic acids. B. cereus may
    overcome these adverse conditions by displaying an adaptive stress response. The response
    of B. cereus upon exposure to these hurdles was investigated using two model strains,
    ATCC 14579 and ATCC 10987. Comparative analysis revealed numerous strain-specific
    genes and differences in metabolic capacities, including a urease encoding gene cluster in
    ATCC 10987 and a nitrate respiration cluster in ATCC 14579. A survey including ATCC
    10987 and 48 environmental and outbreak-associated isolates revealed urease activity, i.e.,
    the conversion of urea in ammonia and carbon dioxide, to be present in 10 isolates.
    However, the activity appeared to be too low to contribute to acid resistance in the strains
    tested. To search for other acid resistance mechanisms, comparative phenotype and
    transcriptome analyses of strains ATCC 14579 and ATCC 10987 cells exposed to organic
    and/or inorganic acid shocks were performed. Upon exposure to low pH with or without the
    addition of lactic acid or acetic acid, common acid resistance mechanisms and induction of
    the nitrate reductase cluster in the more acid resistant strain ATCC 14579 were revealed.
    Furthermore, a major oxidative response was displayed, which included the induction of
    several oxidative stress related genes and the production of inactivation-associated reactive
    oxygen species (ROS), such as hydroxyl radicals, peroxynitrite, and superoxide. ROS were
    detected using fluorescent probes in combination with flow cytometry, including a newly
    developed method using a specific probe that enables superoxide detection in Grampositive
    and Gram-negative bacteria. The formation of ROS was also shown upon exposure
    to heat and was found to be oxygen dependant. Correspondingly, assessment of B. cereus
    stress survival capacity revealed increased heat- and acid-resistance with cells grown and
    exposed to stresses in the absence of oxygen. The excess ROS may originate from stressinduced
    dysfunction of the aerobic electron transfer chain, which was indicated by the
    induction of alternative electron transfer chain components upon exposure to organic and
    inorganic acid shocks. Upon exposure to stress, superoxide is generated through the
    premature leakage of electrons to oxygen at sites in the electron transfer chain at elevated
    rates. Subsequently, superoxide may promote the formation of other ROS, which can cause
    cellular damage leading to cell death. The induction of oxidative stress related genes has
    been reported in numerous other studies involving a wide range of bacteria exposed to
    different adverse conditions. However, a clear relation between the formation of ROS and
    the applied environmental stress was up to now not established. Secondary oxidative responses, including the formation of ROS, are possibly common bacterial responses to
    severe stresses under aerobic conditions. This thesis describes genomic differences between
    B. cereus strains and the acid stress response of these strains on transcriptome and
    phenotype levels, including measurements of intracellular ROS. The findings in this study
    can contribute to further understanding of bacterial stress responses and secondary
    oxidative responses. Furthermore, the results obtained may aid to optimize and select
    (combinations of) stresses to apply in hurdle technology, thus enabling design of safe,
    milder food processing and preservation techniques.
    Two-component signal transduction in Bacillus cereus and closely related bacteria
    Been, M.W.H.J. de - \ 2009
    Wageningen University. Promotor(en): Tjakko Abee; R.J. Siezen, co-promotor(en): C. Francke. - [S.l.] : S.n. - ISBN 9789085854357 - 176
    bacillus cereus - stressreactie - fylogenetica - signaaltransductie - genregulatie - bacillus cereus - stress response - phylogenetics - signal transduction - gene regulation
    Bacillus cereus is a Gram-positive pathogen that is recognised as an important cause of food-borne disease worldwide. Within the genus Bacillus, B. cereus and its closest relatives form a homogeneous subdivision that has been termed the B. cereus group. This group includes B. anthracis, a pathogen that can cause anthrax in mammals, and B. thuringiensis, an insect pathogen that is used as an insecticide worldwide. Members of the B. cereus group can adapt to a wide range of environmental challenges. In bacteria, these challenges are generally monitored by two-component systems (TCS), which consist of a histidine kinase (HK) and a partner response regulator (RR). Upon sensing a specific environmental stimulus, the HK activates its cognate RR, which in turn controls the expression of genes that are involved in the appropriate response. This thesis describes the functional analysis of TCSs in the B. cereus group. By using in silico techniques, 50-58 HKs and 48-52 RRs were identified in eight different B. cereus group genomes. Biological functions, including the involvement in sporulation, biofilm formation and host-microbe interactions were predicted for these TCS proteins. A phylogenetic footprinting approach was developed and used to identify specific binding sites and target genes for over 50% of the B. cereus group DNA-binding RRs. These predictions allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. To further support these predictions, the transcriptomes of two B. cereus TCS deletion mutants (ΔyvrHG and ΔyufLM) were analysed and compared with the transcriptome of wild-type B. cereus. This revealed that the minimal regulon predictions were correct for the two respective TCSs. Furthermore, the predicted biological roles for these TCSs, including roles in antibiotic resistance (YvrHG) and fumarate metabolism (YufLM), were supported by phenotypic tests. Besides the many “classical” HKs and RRs detected in the B. cereus group, several a-typical TCS proteins were found. These included five RRs without a DNA-binding output domain and two hybrid HKs (HK-RR fusions). Genome analyses revealed that one of the hybrid HK-encoding genes (BC1008) is located in a conserved gene cluster that also encodes the a-typical RR RsbY. In B. cereus, RsbY is known to activate the key stress-responsive sigma factor σB. As a partner HK for RsbY was still “missing”, the role of BC1008 in the σB-mediated stress response was tested. Indeed, a bc1008 deletion strain appeared incapable of inducing σB and its associated regulon upon stress conditions and appeared impaired in its heat adaptive response. In addition, truncation of the BC1008 fused RR receiver domain indicated that this domain plays a role in fine-tuning BC1008 activity. A comparative genome analysis further indicated that BC1008-type hybrid HKs control σB-like sigma factors in at least several other Gram-positive bacteria, including Geobacillus, Paenibacillus and actinobacteria. In summary, the research described in this thesis contributes to our understanding of B. cereus adaptive responses through TCSs. This knowledge may be applied for the development of novel intervention strategies for an improved control of B. cereus in food production environments.

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