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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    On the role of vaccine dose and antigenic distance in the transmission dynamics of Highly Pathogenic Avian Influenza (HPAI) H5N1 virus and its selected mutants in vaccinated animals
    Sitaras, Ioannis - \ 2017
    Wageningen University. Promotor(en): M.C.M. Jong, co-promotor(en): B. Peeters. - Wageningen : Wageningen University - ISBN 9789463438063 - 209
    avian influenza viruses - avian influenza - disease transmission - vaccines - vaccination - dosage - antigenic variation - mutants - mutations - immunity - vaccine development - virology - epidemiology - aviaire influenzavirussen - aviaire influenza - ziekteoverdracht - vaccins - vaccinatie - dosering - antigene variatie - mutanten - mutaties - immuniteit - vaccinontwikkeling - virologie - epidemiologie

    Influenza virus infections can cause high morbidity and mortality rates among animals and humans, and result in staggering direct and indirect financial losses amounting to billions of US dollars. Ever since it emerged in 1996 in Guangdong province, People’s Republic of China, one particular highly pathogenic avian influenza (HPAI) H5N1 virus has spread globally, and is responsible for massive losses of poultry, as well as human infections. For these reasons, HPAI H5N1 is considered as one of the viruses possible to cause a future influenza pandemic.

    One of the main reasons why influenza is a recurring problem is its ability to constantly evolve through the selection of mutants that are able to avoid immunity (be it natural or acquired). Due to the accumulation of mutations during genome replication, diverse/variant influenza genome sequences co-exist in a virus pool (quasispecies). These sequences can contain mutations that are able to confer selective advantages to the influenza virus given the opportunity. As a consequence, whenever a situation arises that places the virus under any type of pressure that the dominant virus sequence cannot cope with (i.e. immune pressure, selective receptor binding, etc.), the virus with the genome sequence that allows it to better adapt to that particular pressure becomes selected and takes over.

    Because of the influenza virus’s high rate of mutations, a global surveillance network is in place to monitor changes in circulating strains among humans that would warrant an update of the vaccines used. For human influenza strains, vaccines are updated frequently (every one or two years) and a similar situation holds true for racehorse vaccination. For avian influenza vaccination, however, the situation is different. In most countries, vaccination against avian influenza is not used, and in the countries where vaccines are used (either as routine or emergency measures), they are not updated as frequently as human vaccines are. In addition, in many instances vaccination against avian influenza viruses has met with some spectacular failures, since it failed to produce a level of immunity that would protect against circulating field strains. These vaccination failures have often been attributed to the fact that without constant vaccine updating (as is done for human influenza), the vaccines used are not able to keep up with continuously evolving antigenic variants selected in the field, and thus to protect poultry against them. In addition, since it is known that immune pressure resulting from vaccination can be a driving force in the evolution of influenza viruses and the selection of immune-escape mutants, there is a school of thought that posits that vaccination against avian influenza is not only a very expensive affair (especially if vaccines need to be frequently updated), but can also lead to selection of mutants that are able to avoid vaccination-induced immunity.

    The research reported in this thesis started with addressing the gaps in the knowledge regarding the role of vaccination-induced immunity in the selection of immune-escape mutants of HPAI H5N1, and if there is a way for vaccines to still be able to protect against antigenically-distant variants of the vaccine seed strain, without the need for frequent vaccine updates.

    Our first step in studying influenza virus evolution and selection of immune-escape mutants was to investigate how antigenic pressure may drive the selection of such mutants, and what the effect of the selected mutations on the pathogenicity and transmissibility of the mutants may be. Although there exist a variety of methods to select for influenza virus mutations (i.e. monoclonal antibodies, site-directed mutagenesis, reverse genetics, etc.), none of them is representative of selection as it happens in a vaccinated animal. In Chapter 2, we discuss in detail a laboratory-based system we have developed, in which immune-escape mutants are selected using homologous polyclonal chicken sera, similar to how they are selected in the field due to vaccination- induced immune pressure. We find that selection takes place early on, and additional mutations are selected when immune pressure is increased. Antigenic distances between the selected mutants and their parent strains are also increased throughout the selection process, but not in a linear fashion. Our selection system proved to be robust and replicable, and to be representative of selection in the field, since the mutations we selected for are also found in naturally-selected field isolates, and the antigenic distances between our selected mutants and their parent strains are similar to antigenic distances between vaccine strains and field isolates.

    We continued our research by addressing the roles played by vaccine dose (and resulting immunity) and antigenic distance between vaccine and challenge strains, in the transmission of HPAI H5N1 viruses, by employing transmission experiments using vaccinated chickens (Chapter 3). To our surprise, we found that the effect of antigenic distances between vaccine and challenge strains on transmission is very small compared to the effect of vaccine dose. We then quantified, for the first time, the minimum level of immunity and minimum percentage of the vaccinated population exhibiting said immunity, in order for vaccines to be able to protect against transmission even of strains that are antigenically distant to the vaccine seed strain. Transmission of such strains in well-vaccinated populations would allow for a scenario where vaccination- induced immunity may drive the selection of immune-escape mutants. Our results show that in order for vaccines to prevent transmission of antigenically distant strains (such as the ones resulting from selection due to immune pressure), the threshold level of immunity against these strains should be ≥23 haemagglutination inhibition units (HIU), in at least 86.5% of the vaccinated population. This level of immunity can be estimated by knowing the antigenic distance between the vaccine and challenge (field) strain, and the HI titre against the vaccine strain, which would then allow the approximate level of immunity against the field strain to be deduced. For example, assuming the HI titre against a vaccine strain is 210 HIU, and the distance with the challenge (field) strain is 24 HIU, according to our results the vaccine should be able to protect against the challenge strain, because the difference in HI titres should be around 26 HIU (i.e. above 23 HIU). These results, taken together with our previous work on selection of mutants, where we showed that the antigenic distances between our mutants and their parent strains are representative of distances found in the field, point to the fact that it is unlikely that vaccination-induced immunity can lead to selection of mutants able to escape it, given that a threshold level of immunity in a minimum percentage of the vaccinated population is achieved. As a consequence, we believe that constant vaccine updating may not be necessary for avian influenza viruses, as long as a threshold level of immunity is maintained. This makes vaccination a more attractive control measure, both from a health perspective and a financial one, than just applying biosecurity measures.

    To examine the effect the mutations in the haemagglutinin protein of our selected mutants may have in their transmission among chickens vaccinated with the parent strain, we used reverse genetics techniques to insert the HA gene of our most antigenically distant mutant into the parent strain backbone (Chapter 4). We vaccinated animals with a sub-optimal dose of vaccine, and we concluded that the mutations we selected for did not allow the mutant to avoid even low levels of immunity, such as the ones resulting from a sub-optimal vaccine dose (which resembles a poor field vaccination scenario). At the same time, the HA mutations we selected for did not appear to have a negative effect either on the pathogenicity of the mutant, or its ability to transmit to unvaccinated animals, since both parameters were comparable to the parent strain.

    Finally, we studied the role inter-animal variation in immunity – as measured by HI titres – has in the accuracy of antigenic cartography calculations (Chapter 5). We found that using sera from more than one animal significantly increased the accuracy of antigenic distance calculations, since it takes into account individual differences in immune responses to vaccination, an inevitable phenomenon documented in both humans and animals. In addition, we increased the accuracy of antigenic maps by avoiding the use of dimension-reducing algorithms as is currently done. By not reducing the dimensionality of virus positioning in space, our maps retain the original geometry between strains or sera, leading to more accurate positioning (Chapters 2 and 5). We hope that improving the accuracy of antigenic cartography can lead to a more precise surveillance of influenza evolution and better informed decisions regarding the need to update vaccines.

    Taken collectively, our results can improve field vaccination outcomes, since they provide guidelines on how to increase vaccination efficiency in stopping transmission of even antigenically-distant strains. In addition, our method for selecting for immune- escape mutants can be a valuable addition to research on influenza virus evolution. Moreover, policy making decisions regarding vaccination against any type of influenza can also benefit from our improvement on antigenic cartography accuracy, saving unnecessary costs in vaccine updating, and reducing morbidity and mortality of both animals and humans.

    Antenna size reduction in microalgae mass culture
    Mooij, T. de - \ 2016
    Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Marcel Janssen. - Wageningen : Wageningen University - ISBN 9789462578890 - 196
    algae culture - algae - light - photobioreactors - photosynthesis - mutants - algenteelt - algen - licht - fotobioreactoren - fotosynthese - mutanten

    The thesis describes the potential of microalgae with a reduced light harvesting antenna for biomass production under mass culture conditions (high biomass density, high light intensity). Theoretically, the lower chlorophyll content reduces the light harvesting capacity and with that the amount of photosaturation. The result would be an increase of the biomass yield on light energy, which is especially favorable at high light intensities. In practice, it was found that the productivity of several antenna size mutants strains was equal, or even lower than that of wild type microalgae. The genetically modified algae suffered from a reduced fitness, possibly because the antenna alterations led to impaired photoprotection mechanisms. In an alternative approach, it was found that by spectral tuning (applying different light colours) oversaturation was decreased and the productivity of wild type microalgae was increased. Special attention was paid to photoacclimation behavior of wild type microalgae. It was investigated whether ‘natural acclimation’ can be exploited to maximize productivity. In the last chapter, the competition between antenna size mutants and wild type cells is investigated by means of a modeling approach. It became clear that a wild type infection of an antenna size mutant culture should be prevented at all costs, as the mutants have a reduced competitive strength.

    Strain improvement of oleaginous microalgae
    Jaeger, L. de - \ 2015
    Wageningen University. Promotor(en): Gerrit Eggink; Rene Wijffels, co-promotor(en): Dirk Martens. - Wageningen : Wageningen University - ISBN 9789462574847 - 200
    algen - biomassa - oliën - productiviteit - opbrengsten - transcriptomica - triacylglycerol lipase - bioreactoren - transformatie - mutanten - algenteelt - biomassa productie - algae - biomass - oils - productivity - yields - transcriptomics - triacylglycerol lipase - bioreactors - transformation - mutants - algae culture - biomass production

    The increasing world population and living standards have enlarged the demand for food, feed, and for chemicals. Traditional fossil fuel based commodities need to be replaced, not only because these resources are finite, but also to relieve the impact of carbon emission and pollution, resulting from fossil fuel derived processes. Much attention is on using plants to produce sustainable, renewable alternatives to petrochemical based processes. Palm oil is the crop with the highest lipid yield known today, but the production of palm oil causes deforestation on a large scale. Microalgae are a promising platform for the production of sustainable commodity products. A commodity product that can be produced in microalgae is triacylglycerol (TAG). The TAG molecules that are accumulated in microalgae are comparable to the TAG profiles of commonly used vegetable oils, and can directly be applied for edible oil as well as for biodiesel production. Currently, microalgae derived products have proven to be functional and a potential replacement for conventional crops. However, microalgae derived products, especially TAGs, are not economically feasible yet. In order to make microalgal derived products a reality we need to decrease the production costs by smart technological solutions, biological understanding and metabolic engineering.

    To get more insight in the lipid accumulation mechanism of microalgae, and to define targets for future strain improvement strategies, transcriptome sequencing of the oleaginous microalgae Neochloris oleoabundans was done. This oleaginous microalga can be cultivated in fresh water as well as salt water. The possibility to use salt water gives opportunities for reducing production costs and fresh water footprint for large scale cultivation.

    In chapter 2 the lipid accumulation pathway was studied to gain insight in the gene regulation 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen depleted conditions in a comparable way in both fresh and salt water. The transcriptome sequencing revealed a number of genes, such as glycerol-3-phosphate acyltransferase and via glycerol-3-phosphate dehydrogenase, that are of special interest and can be targeted to increase TAG accumulation in microalgae. NMR spectroscopy revealed an increase in proline content in saline adapted cells, which was supported by up regulation of the genes involved in proline biosynthesis. In addition to proline, the ascorbate-glutathione cycle seems to be of importance for successful osmoregulation by removal of reactive oxygen species in N. oleoabundans, because multiple genes in this pathway were upregulated under salt conditions. The mechanism behind the biosynthesis of compatible osmolytes in N. oleoabundans can be used to improve salt resistance in other industrially relevant microalgal strains.

    Another very promising candidate for TAG production is the oleaginous green microalga Scenedesmus obliquus.

    In chapter 3, UV mutagenesis was used to create starchless mutants, since no transformation approach was available for this species, due to its rigid and robust cell wall. All five starchless mutants that were isolated from over 3500 screened mutants, showed an increased triacylglycerol productivity. All five starchless mutants showed a decreased or completely absent starch content. In parallel, an increased TAG accumulation rate was observed for the starchless mutants and no substantial decrease in biomass productivity was perceived. The most promising mutant (Slm1) showed an increase in TFA productivity of 41% at 4 days after nitrogen depletion and reached a TAG content of 49.4% (%CDW).

    In chapter 4 the Slm1 strain was compared to the wild type strain using photobioreactors. In the wild type, TAG and starch accumulated simultaneously during initial nitrogen starvation, and starch was subsequently degraded and likely converted into TAG. The Slm1 did not produce starch and the carbon and energy acquired from photosynthesis was partitioned towards TAG synthesis. This resulted in an increase of the maximum TAG content in Slm1 to 57% (%CDW) compared to 45% (%CDW) in the wild type. Furthermore, it increased the maximum yield of TAG on light by 51%, from 0.144 in the wild type to 0.217 g TAG mol-1 photon-1 in the Slm1 mutant. No differences in photosynthetic efficiency between the Slm1 mutant and the wild type were observed, indicating that the mutation specifically improved carbon partitioning towards TAG and the photosynthetic capacity was not affected.

    To identify the mutation that caused the starchless phenotype of Slm1 the transcriptome of both the wild type and the Slm1 mutant was sequenced as described in chapter 5. A single nucleotide polymorphism (SNP) was discovered in the small subunit of the starch biosynthesis rate-controlling enzyme ADP-glucose pyrophosphorylase, which resulted in the introduction of a STOP codon in the messenger RNA of the enzyme. The characterization of the mutation increases the understanding of carbon partitioning in oleaginous microalgae, leading to a promising target for future genetic engineering approaches to increase TAG accumulation in microalgae.

    To use the insight that is gained in chapters 2-5 for metabolic engineering of TAG accumulation and carbon partitioning, a metabolic engineering toolbox is required. However, the development of transformation protocols for new and less well studied industrially relevant microalgae is challenging. In chapter 6, a simple and effective tool for the optimization of transformation protocols is proposed. Optimal voltage settings were determined for five microalgae: C. reinhardtii, Chlorella vulgaris, N. oleoabundans, S. obliquus, and Nannochloropsis sp. This method can be used to speed up the screening process for species that are susceptible for transformation and to successfully develop transformation strategies for industrially relevant microalgae, which lack an efficient transformation protocol.

    In addition to the increase in productivity, improving the quality in terms of fatty acid composition of TAG molecules would be desired as well. For example, the accumulation of stearic acid rich TAG molecules is of special interest, because of the improved structural properties. The lipid accumulating starchless mutant of the model species C. reinhardtii BAFJ5 was used as model species in chapter 7, since genetic toolbox is well established for this species. In this chapter, stearoyl-ACP desaturase (SAD), is silenced by artificial microRNA. The mRNA levels for SAD were reduced after the silencing construct was induced. In one of the strains, the reduction in SAD mRNA resulted in a doubling of the stearic acid content in triacylglycerol molecules, which shows that increasing the fraction of stearic acid in TAG is possible. Furthermore, we hypothesize that in addition to direct conversion in the chloroplast, C. reinhardtii is able to redirect stearic acid from the chloroplast to the cytosol and convert it to oleic acid in the endoplasmic reticulum by stearoyl-CoA desaturase.

    In chapter 8, an outlook is given on microalgal strain improvement strategies for the future, reflecting on the results obtained in this thesis. Also a roadmap is suggested to get genetically modified microalgal derived products on the market. The results presented in this thesis, provide a significant improvement in the understanding of TAG accumulation and carbon partitioning in oleaginous microalgae. Furthermore, improved microalgal strains with increased TAG accumulation or improved TAG fatty acid composition under nitrogen depleted conditions were generated. In addition, an outlook is presented in which the major bottlenecks are presented in future industrial applications of microalgae.

    Regulation and natural functions of lipopeptide biosynthesis in Pseudomonas
    Song, C. - \ 2015
    Wageningen University. Promotor(en): Francine Govers, co-promotor(en): Jos Raaijmakers. - Wageningen : Wageningen University - ISBN 9789462572690 - 173
    pseudomonas fluorescens - lipoproteïnen - biosynthese - genetische kartering - genregulatie - genomica - transcriptomica - verdedigingsmechanismen - protozoa - mutanten - pseudomonas fluorescens - lipoproteins - biosynthesis - genetic mapping - gene regulation - genomics - transcriptomics - defence mechanisms - protozoa - mutants


    Lipopeptides (LPs) are surface-active, antimicrobial compounds composed of a lipid moiety linked to a short linear or cyclic oligopeptide. In bacteria, LPs are synthesized by large nonribosomal peptide synthetases (NRPSs) via a thiotemplate process. Compared to the understanding of LP biosynthesis, little is known about the genetic regulation.

    The aims of this PhD thesis were to identify new regulatory genes of LP biosynthesis and to unravel the natural functions of LPs in plant-associated Pseudomonas species. Using a combination of various ‘omics’-based technologies, we identified two small RNAs, designated RsmY and RsmZ, that, together with the repressor proteins RsmA and RsmE, regulate the biosynthesis of the LP massetolide in the rhizosphere bacterium Pseudomonas fluorescens SS101. Four other regulatory genes (phgdh, dnaK, prtR and clpA) of massetolide biosynthesis were identified via random mutagenesis. Mutations in each of these four genes caused a deficiency in massetolide production, swarming motility and biofilm formation, two natural functions associated with the production of LPs in Pseudomonas. Results further indicated that the ClpAP protease complex regulates massetolide biosynthesis via the pathway-specific, LuxR-type regulator MassAR, the heat shock proteins DnaK and DnaJ, and proteins of the TCA cycle.

    LPs exhibit broad-spectrum antimicrobial activities and have diverse natural functions for the producing bacteria. LPs of P. fluorescens were shown to play an important role in defense against protozoan predation. Genome-wide transcriptome analysis revealed that 55 and 73 genes were up- and down-regulated respectively in P. fluorescens strain SS101 upon grazing by the protozoan predator Naeglaria americana. The up-regulated genes included the LP biosynthesis genes massABC, but also genes involved in alkane degradation and in putrescine catalysis. Putrescine induced encystment of the protozoa, possibly providing a second line of defense against predation. MALDI imaging mass spectrometry (IMS) and live colony NanoDesi mass spectrometry further revealed, in real time, site-specific LP production at the interface of Pseudomonas-protozoa interactions. When the closely related strain P. fluorescens SBW25 was exposed to N. americana, similar overall transcriptional and metabolic responses were observed as found for strain SS101, but also strain-specific responses were apparent. These results indicate that closely related bacterial strains exhibit common and unique transcriptomic and metabolic responses to protozoan predation. Next to defense against competitors and predators, LPs are well-known for their role in swarming motility, a flagella-driven multicellular behavior of bacteria. Orfamide-deficient mutants of P. protegens Pf-5, either with deletions in the biosynthesis gene ofaA or in the regulatory gene gacA, cannot swarm on their own but ‘hitch-hike’ with parental strain Pf-5. However, distinctly different spatial distributions in co-swarming colonies were observed for these two mutants, with the ofaA mutant moving behind the wild type and the gacA mutant predominating on the edge of the swarming colony. Subsequent experimental evolution assays showed that repeated swarming cycles of strain Pf-5 drives parallel evolution toward fixation of spontaneous gacS/gacA mutants on the edge, ultimately causing colony collapse. Transcriptome analyses revealed that genes associated with resource acquisition, motility, chemotaxis and efflux were significantly upregulated in these regulatory mutants. Moreover, microscopic analysis showed that gacA mutant cells were longer and more flagellated than wild type and ofaA mutant cells, which may explain their predominance on the edge of co-swarming colonies. Collectively, these results indicated that adaptive convergent evolution through point mutations is a common feature of range-expanding microbial populations and that the putative fitness benefits of these spontaneous mutations during dispersal of bacteria into new territories are frequency-dependent.

    Elstar mutanten (poster)
    Heijerman-Peppelman, G. ; Elk, P.J.H. van - \ 2014
    fruitgewassen - appels - mutanten - gebruikswaarde - kwaliteitsnormen - hagelbescherming - fruit crops - apples - mutants - use value - quality standards - hail protection
    De consument herkent Elstar niet meer vanwege de grote hoeveelheid kleurmutanten. Daarom doet PPO onafhankelijk onderzoek naar de gebruikswaarde van acht verschillende mutanten om te komen tot uniforme Elstar van hoge kwaliteit in het winkelschap. De belangrijkste teelteigenschappen bij aanplant met- en zonder hagelnetten worden in beeld gebracht.
    Bestrijding vroegtijdige bladvalziekte bij Golden Delicious mutanten in de boomkwekerij
    Wenneker, M. ; Bruine, J.A. de - \ 2014
    Randwijk : Praktijkonderzoek Plant & Omgeving, Business Unit Bloembollen, Boomkwekerij en Fruit - 27
    malus - rassen (planten) - bladval - mutanten - aantasting - symptomen - proeven - detectie - bestrijdingsmethoden - vruchtbomen - malus - varieties - leaf fall - mutants - infestation - symptoms - trials - detection - control methods - fruit trees
    Vroegtijdige bladval bij Golden Delicious (mutanten) is een fenomeen dat wereldwijd optreedt. In de jaren 1960-1970 is voor dit probleem veel aandacht geweest in Nederland. Hierbij is gekeken naar voeding, weersinvloeden en diverse ziekteverwekkers, maar tot een oplossing heeft dit niet geleid. De bladval werd uiteindelijk aanvaard als iets wat bij het ras hoorde. De problematiek van vroegtijdige bladval in de vruchtboomkwekerij was aanleiding om nieuw onderzoek te starten. Het ras Golden Delicious is in Nederland minder belangrijk geworden. Het fenomeen vroegtijdige bladval bestaat echter nog steeds. Vaak worden bladmeststoffen gespoten om het probleem, meestal zonder succes, tegen te gaan. Vruchtboomkwekers ervaren kwaliteitsverlies door vroegtijdige bladval bij Golden. De symptomen in de kwekerij en de boomgaard zijn vergelijkbaar. Eerst ontstaan necrotische vlekjes op het blad, dan vergelen de bladeren en tegelijkertijd begint de vroegtijdige bladval. In de vruchtboomkwekerij resulteert deze bladval in verkaling van het hout en bomen van lichtere kwaliteit. In de fruitteelt is bladval bij Golden Delicious ook nog steeds een probleem. Daar kan het de kwaliteit van de vruchten nadelig beïnvloeden.
    Identification of genes affecting the response of tomato and Arabidopsis upon powdery mildew infection
    Gao, D. - \ 2014
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Yuling Bai; Anne-Marie Wolters. - Wageningen : Wageningen University - ISBN 9789462570122 - 144
    solanum lycopersicum - tomaten - arabidopsis thaliana - plantenziekteverwekkende schimmels - oidium neolycopersici - genen - ziekteresistentie - wilde verwanten - mutanten - genexpressie - plantenveredeling - solanum lycopersicum - tomatoes - arabidopsis thaliana - plant pathogenic fungi - oidium neolycopersici - genes - disease resistance - wild relatives - mutants - gene expression - plant breeding

    Many plant species are hosts of powdery mildew fungi, including Arabidopsis and economically important crops such as wheat, barley and tomato. Resistance has been explored using induced mutagenesis and natural variation in the plant species. The isolated genes encompass loss-of-function susceptibility genes and dominantly inherited genes encoding NB-LRR proteins, receptor-like kinases or proteins that do not have typical resistance protein domains. Cultivated tomato is susceptible to powdery mildew species Oidium neolycopersici, and exploiting the resistance genes present in wild tomato species is a favourable strategy to control the disease. In chapter 2, we give an overview of all the identified resistance genes in wild tomato species and their resistance mechanisms inferred from cytological and molecular data. Furthermore, resistance genes and their mechanisms are compared between tomato and other plant species, such as dicot Arabidopsis and monocots barley and wheat. This comparison illustrates that both common and species-specific mechanisms are involved with respect to resistance to powdery mildews in different plant species.

    Resistance gene Ol-1 originates from wild tomato species S. habrochaites. It confers race-non-specific resistance to tomato powdery mildew. To elucidate the resistance signalling pathway, we adopted a virus induced gene silencing (VIGS) approach to suppress genes which are differentially expressed when comparing genotypes with and without the Ol-1 introgression. In chapter 3, we showed that ALS (acetolactate synthase) activity is important for Ol-1-mediated resistance, as simultaneous silencing of two ALS genes attenuated the resistance level of NIL-Ol-1. ALS is a key enzyme in the biosynthesis of branched-chain amino acids, and a target of commercial herbicides. Reducing ALS activity via herbicidal treatment did not result in altered responses to powdery mildew infection in susceptible cultivar Moneymaker and resistant line NIL-Ol-4, indicating that ALS is not involved in basal defense nor in NB-LRR gene-mediated resistance. Whether the role of ALS in Ol-1-mediated resistance is associated with amino acid homeostasis is unknown and needs further investigation.

    Besides tomato, Arabidopsis is a host of powdery mildew O. neolycopersici. The large collection of Arabidopsis accessions and several mutant collections are valuable resources to identify novel resistance genes. In chapter 4, we first screened 123 Arabidopsis accessions for O. neolycopersici resistance and then studied the genetic basis of theresistance by segregation analysis in 19 F2 populations. The results showed that polygenic resistance is the main form of resistance. Accession C24 displays complete resistance with polygenic nature, as shown by QTL analysis of the F2 population derived from the cross between C24 and susceptible accession Sha. The recessively inherited locus on chromosome 1 was fine-mapped by recombinant screening, and analysis of candidate genes resulted in the isolation of the gene conferring resistance. It proved to be a mutant allele of EDR1, harbouring a deletion upstream of the kinase domain resulting in a truncated protein. Previously, an induced edr1mutationin Col-0 background was obtained. However, the edr1 mutation in our C24 source (referred to as C24-W) occurred in a different position. The resistance conferred by edr1 in C24-W was not associated with constitutively expressed pathogenesis-related genes. Remarkably, we observed that although C24-W carried the edr1 mutation this mutation was absent in other C24 sources. In addition, C24-W was smaller in size than C24 from other sources. Since the edr1 mutation confers resistance to tomato powdery mildew in Arabidopsis, we investigated whether this resistance system is conserved in tomato. The results showed that individual silencing of two tomato EDR1 candidate genes in susceptible cultivar Moneymaker did not result in decreased sporulation of tomato powdery mildew.

    In chapter 5, we screened an activation tag Arabidopsis mutant collection. In these mutants, tagged genes are overexpressed by the strong 35S enhancers resulting in a dominant gain-of-function phenotype. One mutant line, 3221, was identified due to its resistance to powdery mildew O. neolycopersici. Additional disease tests showed that 3221 displayed resistance to the downy mildew Hyaloperonospora arabidopsidis and the aphid Myzus persicae, but susceptibility to the bacterial pathogen Pseudomonas syringae pv tomato DC3000. The mutant line 3221 also showed reduced size and serrated leaves, and the altered morphology was associated with resistance. Inverse PCR and expression analysis revealed that the gene underlying the resistance was ATHB13, a HD-Zip transcription factor. Suppression ofATHB13 in 3221 by RNAi transformation resulted in the loss of resistance and altered morphology, while overexpression of ATHB13 in wild-type plants induced resistance and altered morphology. Microarray analysis of 3221 and the parental line Ws resulted in the identification of a large number of genes showing differential expression. Analysis of these results did not give a clear indication that the resistance phenotype in 3221 is due to the activation of classical hormone pathway genes involved in resistance. The possibility of utilizing ATHB13 for engineering pathogen resistance in tomato needs to be investigated in the future.

    Finally, in chapter 6 the results from the previous chapters are discussed in a broader context.

    Towards generating broad-spectrum resistance to pathogens in plants: studies on a down-stream signalling NB-LRR of tomato
    Sueldo, D.J. - \ 2014
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Matthieu Joosten; Wladimir Tameling. - Wageningen : Wageningen University - ISBN 9789461738974 - 209
    solanum lycopersicum - tomaten - ziekteresistentie - verdedigingsmechanismen - receptoren - pathogenesis-gerelateerde eiwitten - bindende eiwitten - virulentie - mutanten - genetische kartering - solanum lycopersicum - tomatoes - disease resistance - defence mechanisms - receptors - pathogenesis-related proteins - binding proteins - virulence - mutants - genetic mapping
    Elstar mutanten onder hagelnet later rijp (poster)
    Heijerman-Peppelman, G. ; Elk, P.J.H. van; Dieren, M.C.A. van - \ 2013
    malus - rassen (planten) - cultivars - mutanten - kleur - gebruikswaarde - gewaskwaliteit - consumenten - hagelbescherming - landbouwkundig onderzoek - malus - varieties - cultivars - mutants - colour - use value - crop quality - consumers - hail protection - agricultural research
    De consument herkent Elstar niet meer vanwege de grote hoeveelheid kleurmutanten. Daarom doet PPO onafhankelijk onderzoek naar de gebruikswaarde van acht verschillende mutanten om te komen tot uniforme Elstar van hoge kwaliteit in het winkelschap. De belangrijkste teelteigenschappen bij aanplant met en zonder hagelnetten worden in beeld gebracht.
    Identification of Arabidopsis thaliana genes that can increase resistance towards phloem feeding insects
    Chen, X. - \ 2013
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Ben Vosman. - [S.l.] : S.n. - ISBN 9789461737649 - 96
    arabidopsis thaliana - insectenplagen - myzus persicae - plaagresistentie - genkartering - genexpressie - mutanten - plantenveredeling - turnip yellows virus - vectoren, ziekten - arabidopsis thaliana - insect pests - myzus persicae - pest resistance - gene mapping - gene expression - mutants - plant breeding - turnip yellows virus - disease vectors

    Phloem feeding insects are among the most devastating pests worldwide. They not only cause damage by feeding from the phloem, but also by vectoring plant viruses. During their evolution plants have developed a variety of defense traits to combat insects. These plant resistance traits can be antixenotic and/or antibiotic. Antixenosis is the first line of defense that prevents insects from landing and settling, while antibiosis reduces the population development of the colonizing insects.In this project we aimed at identifying genes that can increase resistance towards phloem feeding insects and also prevent, as far as possible, transmission of viruses. Acknowledging that changing the expression level or expression localization of genes might increase resistance, we screened an Arabidopsis thaliana activation tag gain-of-function mutant collection for increased resistance towards the green peach aphid (Myzus persicae). In these mutants, tagged genes are overexpressed by the strong 35S enhancer adjacent to the natural promoter that results in a dominant gain-of-function phenotype. The overexpression of a particular gene in such mutants may result in enhanced resistance to aphids and other phloem feeding insects.

    To identify mutants with increased insect resistance efficient and reproducible screening methods needed to be developed first. Based on the hypothesis that there is a trade-off between plant fitness and plant resistance, we first screened a subset of 170 mutants that were previously selected based on their reduced growth to increase the chance of identifying mutants with increasedresistance. In this screening we usedchoice assays and selected one mutant that displays enhanced antixenosis based resistance towards aphids. Further characterization of this mutant revealed that that the antixenosis is phloem based and requires intact plants.

    To evaluate aphid resistance of a larger number (>5000) of activation tag mutants, we established a high throughput screening system in which plant resistance against aphids is inferred from a reduced transmission of the circulative Turnip yellows virus(TuYV). This virus can only be transmitted into a plant after virus-infected aphids feed for a prolonged (> 10min) time from the phloem sap. In the initial screening 13 virus-free mutant lines were identified. The putative candidate mutant lines were re-evaluated and characterized, resulting in nine mutants on which aphids showed a reduced population development.

    Molecular analysis of two of these mutants revealed that the genes underlying the resistance were IRM1(Increased ResistancetoMyzus persicae1,At5g65040)and SKS13 (SKU5Similar13, At3g13400). In wild type plants,IRM1is strongly expressed in xylem and extremely low expressed in other plant tissue whereas SKS13 is exclusively expressed in pollen. We show that constitutive overexpression of these genes in all plant tissues confers enhanced resistance towards aphids. Analysis of aphid feeding behavior showed that the resistance conferred by IRM1and SKS13affect the aphids differently. On the IRM1 overexpressing mutant aphids encounter difficulties in reaching the phloem, indicating that resistance factors are located between the cell surface and the phloem. On the SKS13overexpressingmutant the phloem feeding of aphids is severely affected, indicating that resistance factors are phloem based. Further analysis strongly suggests the involvement of Reactive Oxygen Species (ROS) in the reduced aphid performance on the SKS13overexpressingmutant. We also show that the resistances are not aphid specific, as the performance of the cabbage aphid (Brevicoryne brassicae)is also affectedon both overexpressing mutants.

    The results obtained in this thesis show that plant resistance to insects can be increased by expressing genes that are assigned for other biological functions. Characterization of the identified mutants revealed twogenes conferring enhanced aphid resistance via different mechanisms. These findings lead to a better understanding of plant-aphid interactions on the molecular level. Furthermore, such knowledge obtained from the model plant A.thalianashould be applied in crop plants, which can be achieved by transgenic and genetic studies in combination with newly developed techniques, such as RNAi and TILLING.

    Bladval voorkomen met Alternaria-middel
    Wenneker, M. - \ 2013
    De Fruitteelt 103 (2013)8. - ISSN 0016-2302 - p. 15 - 15.
    malus - rassen (planten) - bladval - plantenplagen - mutanten - landbouwkundig onderzoek - oorzakelijkheid - bemesting - varieties - leaf fall - plant pests - mutants - agricultural research - causality - fertilizer application
    Vroegtijdige bladval bij Golden Delicious (mutanten) is een wereldwijd fenomeen. In de jaren 1960-1970 was er veel aandacht voor in Nederland. Men keek naar voeding, weersinvloeden en diverse ziekteverwekkers, maar tot een oplossing leidde dit niet. De bladval werd uiteindelijk aanvaard als iets wat bij het ras hoorde. De vroegtijdige bladval in de vruchtboomkwekerij was aanleiding om een nieuw onderzoek op te zetten.
    Jubileum voor de zandraket
    Sikkema, A. - \ 2010
    Resource: weekblad voor Wageningen UR 4 (2010)8. - ISSN 1874-3625 - p. 18 - 20.
    arabidopsis thaliana - mutanten - genomen - genetische bronnen - genetische modellen - plantenfysiologie - wetenschappelijk onderzoek - arabidopsis thaliana - mutants - genomes - genetic resources - genetic models - plant physiology - scientific research
    Je kunt ’m in Nederland op elke straathoek tegenkomen tussen de trottoirtegels: de zandraket of Arabidopsis thaliana. Dit jaar viert dit ‘onkruid’ zijn zilveren jubileum als modelplant van de plantenwetenschappers. Onderzoek aan dit plantje heeft geleid tot detailkennis van vrijwel alle moleculaire processen in planten.
    Genome-wide investigation into roles of Arabidosis receptor-like proteins in pathogen defense
    Ellendorff, U. - \ 2009
    Wageningen University. Promotor(en): Pierre de Wit, co-promotor(en): Bart Thomma. - [S.l. : S.n. - ISBN 9789085853206 - 141
    planten - arabidopsis thaliana - verdedigingsmechanismen - plantenziekteverwekkers - planteiwitten - pathogenesis-gerelateerde eiwitten - genoomanalyse - mutanten - genexpressie - plant-microbe interacties - gene silencing - plants - arabidopsis thaliana - defence mechanisms - plant pathogens - plant proteins - pathogenesis-related proteins - genome analysis - mutants - gene expression - plant-microbe interactions - gene silencing
    Receptor-like proteins (RLPs) are receptors on the surface of plant cells that are important for the activation of disease resistance. Furthermore, some RLPs are important for plant development. The Arabidopsis genome contains 57 genes encoding RLPs. A genome wide collection of RLP gene knock-out mutants was assembled and functionally analyzed for defects in defense and development. This resulted in the identification of an RLP that plays a role in hormone perception, and two RLPs that play a role in non-host resistance, the phenomenon that a plant species is typically resistant to pathogens of other plant species.
    RNA silencing is a regulatory mechanism by which the expression of genes is downregulated or entirely suppressed. In this thesis, it is demonstrated for the first time that this mechanism is important for defense of Arabidopsis against a fungal pathogen; the vascular wilt fungus Verticillium. This is an extremely important pathogen of over 200 plant species including economically important crops.

    Corina, een vroege mutant van Conference
    Meijer, H. ; Dieren, M.C.A. van - \ 2009
    De Fruitteelt 99 (2009)4. - ISSN 0016-2302 - p. 13 - 13.
    fruitteelt - peren - rassen (planten) - mutanten - fruit growing - pears - varieties - mutants
    Corina, rasnaam Saels, is een stabiele, vroege mutant van het perenras Conference en werd gevonden door de gebroeders Saels in België. Samen met de Belgische boomkwekerij René Nicolaï hebben zij een concept voor marktintroductie en afzet ontwikkeld
    Niet-waardplant resistentie tegen Phytophthora infestans in modelplant Arabidopsis thaliana
    Vossen, E.A.G. van der; Arkel, G. van - \ 2008
    phytophthora infestans - arabidopsis - mutanten - arabidopsis thaliana - genetische modificatie - plaagresistentie - vatbaarheid - phytophthora infestans - arabidopsis - mutants - arabidopsis thaliana - genetic engineering - pest resistance - susceptibility
    In een verzameling van ca. 5.000 Arabidopsis deletielijnen wordt gezocht naar mutanten die een zeldzame vatbaarheid vertonen voor P. infestans
    Experimental ecology and evolution of microbial diversity : the role of spatial structure
    Habets, M.G.J.L. - \ 2008
    Wageningen University. Promotor(en): Rolf Hoekstra, co-promotor(en): Arjan de Visser. - S.l. : s.n. - ISBN 9789085048619 - 102
    micro-organismen - diversiteit - biodiversiteit - evolutie - ecologie - adaptatie - heterogeniteit - mutanten - microbiële diversiteit - microorganisms - diversity - biodiversity - evolution - ecology - adaptation - heterogeneity - mutants - microbial diversity
    In the light of the competitive exclusion principle, which states that complete competitors cannot coexist, many explanations have been sought to explain the high diversity found in nature. The most common explanation is the niche differentiation hypothesis: coexistence is obtained through differentiation of species in ecological niches. Spatial structure is thought to be a factor capable of providing opportunities for niche differentiation. We have focused on four aspects of spatial structure enabling genetic diversity to emerge and /or to be maintained.
    First of all, population fragmentation, resulting from growth in spatially structured habitats, can increase diversity, because the resulting smaller subpopulations, due to their smaller population size, are more likely to adaptively diverge. By allowing small and large populations of E. coli to evolve for 500 generations in two different nutrient environments, we test this hypothesis. The results demonstrate higher variance in fitness among small populations, and consequently more heterogeneous adaptive trajectories for small populations, some of which surprisingly lead to higher fitness peaks than reached by even the best adapted large population.
    In a short-term invasion experiment between a superior E. coli competitor and its inferior ancestor, we demonstrate that populations residing in structured environments experience slower invasion dynamics of beneficial mutations than well-mixed populations due to limited dispersal, and therefore local competition. Moreover, our results demonstrate a deceleration of invasion with increasing size of the invading subpopulation. This is caused by a decrease of inter specific competition relative to intra specific competition. Since inferior competitors are present in the community for a longer period of time, they can recombine with other persisting lineages or obtain new mutations, some of which might be beneficial. It is therefore possible that polymorphisms arise which would not have had the opportunity to emerge in a well-mixed environment. Even though both population fragmentation and slower competitive dynamics can increase the emergence of diversity, they do not provide a means for their maintenance.
    Environmental heterogeneity on the other hand can cause maintenance of diversity. Environmental heterogeneity can be introduced by spatial structure, e.g. by providing gradients in biotic and abiotic factors, thereby increasing the number of niches. By allowing E. coli populations to evolve for 900 generations in either a well-mixed environment or two structured environments (with or without dispersal), we demonstrate stable coexistence of diversity in structured populations without dispersal. This can be attributed to negative frequency-dependent fitness interactions among niche specialists that either inhabit existing niches provided by the heterogeneous environment or new niches constructed by organisms inhabiting the environment.
    In addition to examining aspects of spatial structure that provide means for populations to diversify, we examine a specific consequence of slower dynamics and environmental heterogeneity: the probability of mutators to hitchhike to fixation. Understanding the emergence of mutators is not only scientifically important, but also relevant for human health, since high frequencies of mutators have been found in bacterial populations and drug resistant mutants arise more often in mutator populations. E. coli mutator populations were introduced at different starting frequencies in a well-mixed environment and two structured environments differing in their dispersal rate. Contrary to expectations, we find an advantage in the rate of invasion for mutators in well-mixed environments. Faster competitive dynamics may allow a rapid increase of population size and hence a greater supply of mutations for subsequent adaptation. Due to a delay in mutator extinction in structured environments at low frequencies, mutators may gain from fluctuating conditions.

    Vossen, E.A.G. van der; Loonen, A.E.H.M. - \ 2007
    solanum - phytophthora - ziekteresistentie - mutanten - genexpressie - solanum - phytophthora - disease resistance - mutants - gene expression
    In dit project wordt onderzocht in hoeverre R-genen in de plant verschillende werkingsmechanismen hebben
    Molecular analysis of plant architecture in Arabidopsis thaliana using activation tagging.
    Chalfun Junior, A. - \ 2004
    Wageningen University. Promotor(en): Maarten Koornneef; Gerco Angenent. - Wageningen : S.n. - ISBN 9789085040361 - 144
    arabidopsis thaliana - transposons - dna - mutanten - plantenontwikkeling - plantenmorfologie - moleculaire genetica - genexpressie - arabidopsis thaliana - transposable elements - dna - mutants - plant development - plant morphology - molecular genetics - gene expression
    Keywords: Arabidopsisthaliana, activation tagging, T-DNA, transposon, mutants, enhancer, DNA methylation, plant architecture, development, forward/reverse genetics, lateral organs, flower, vascular tissue, HLH, transmembrane, transcription factors

    Plant development is one of the most important aspects of plant's life cycle that has extensively been studied at the morphological, genetic and molecular level. It is import for systematic and taxonomic classification, but also for applied agronomic reasons, because it affects the growth and cultivation leading to higher yield and quality of the product.The generation of genetic variants, like mutants may increase genetic pool and gives information about plant processes and their genetic control.Activation tagging is a new powerful tool to generate and identify new mutants, which emerged as an alternative for gene function analysis. This thesis reports the study on the molecular control of plant architecture, using mutants generated by an activation tagging-based approach in the model plant Arabidopsis thaliana . In addition, it also describes experiments that could explain why the low frequencies of mutants were obtained by T-DNA based activation tagging. Based on this comparison, the transposon-based activation tagging strategy was chosen and a screen for flower and silique mutants in a large Arabidopsis population yielded three gain-of-function mutants. These mutants were designated downwards siliques1 ( ds1-D ), needle1 ( ndl1-D ) and twisted1 ( twt1-D ). In the ds1-D mutant, internodes are shorter and the lateral organs such as flowers are bending downwards. Further molecular and genetic studies on this mutant revealed that DS1 is important to control petiole-blade boundary in Arabidopsis petals. In the ndl1-D mutant, the normal formation of valve tissues is altered, resulting in a pin-like structure that replaces the two fused carpels of the wild type pistil. The results suggest that NDL1 is involved in normal carpel development, in which auxin distribution plays an important role. In the third mutant, twt1-D , the overexpression of TWT1 led to twisting of all organs, whichismost pronounced in siliques. This phenotype and the expression pattern of the gene suggest that TWT1 is involved in proper vascular tissue development in Arabidopsis . These studies demonstrate the power of activation tagging and it gains valuable knowledge about the molecular networks that control plant development.
    The genetics of seed quality in Arabidopsis thaliana
    Clerkx, E.J.M. - \ 2004
    Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Steven Groot. - Wageningen : S.n. - ISBN 9789058089700 - 136
    zaadkwaliteit - genetische regulatie - zaadlevensduur - droogteresistentie - genetische variatie - genen - mutanten - seed quality - genetic regulation - seed longevity - drought resistance - genetic variation - genes - mutants
    Verslag Boskoopmutantenproef Horst en Wilhelminadorp : 069-9502
    Steeg, P.A.H. van der; Kemp, H. - \ 2002
    Randwijk : Praktijkonderzoek Plant & Omgeving, Sector Fruit (Rapport / Praktijkonderzoek Plant & Omgeving, Sector Fruit nr. 2002-1) - 43
    appels - mutanten - gebruikswaarde - kwaliteit - plantenveredeling - apples - mutants - use value - quality - plant breeding
    Dit onderzoek is gefinacierd door het productschap Tuinbouw
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