Staff Publications

Staff Publications

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

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

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

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    Revisiting the Role of Master Regulators in Tomato Ripening
    Wang, Rufang ; Angenent, Gerco C. ; Seymour, Graham ; Maagd, Ruud A. de - \ 2020
    Trends in Plant Science 25 (2020)3. - ISSN 1360-1385 - p. 291 - 301.
    CRISPR- mutagenesis - gain-of-function - mutants - ripening - tomato - transcription factors

    The study of transcriptional regulation of tomato ripening has been led by spontaneous mutations in transcription factor (TF) genes that completely inhibit normal ripening, suggesting that they are ‘master regulators’. Studies using CRISPR/Cas9 mutagenesis to produce knockouts of the underlying genes indicate a different picture, suggesting that the regulation is more robust than previously thought. This requires us to revisit our model of the regulation of ripening and replace it with one involving a network of partially redundant components. At the same time, the fast rise of CRISPR/Cas mutagenesis, resulting in unexpectedly weak phenotypes, compared with knockdown technology, suggests that compensatory mechanisms may obscure protein functions. This emphasises the need for assessment of these mechanisms in plants and for the careful design of mutagenesis experiments.

    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

    Summary

    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.

    REDUCED DORMANCY5 Encodes a Protein Phosphatase 2C That Is Required for Seed Dormancy in Arabidopsis
    Xiang, Y. ; Nakabayashi, K. ; Ding, J. ; He, F. ; Bentsink, L. ; Soppe, W.J.J. - \ 2014
    The Plant Cell 26 (2014)11. - ISSN 1040-4651 - p. 4362 - 4375.
    rna-binding proteins - abscisic-acid - messenger-rna - pp2c phosphatases - germination - thaliana - aba - reveals - gene - mutants
    Seed dormancy determines germination timing and contributes to crop production and the adaptation of natural populations to their environment. Our knowledge about its regulation is limited. In a mutagenesis screen of a highly dormant Arabidopsis thaliana line, the reduced dormancy5 (rdo5) mutant was isolated based on its strongly reduced seed dormancy. Cloning of RDO5 showed that it encodes a PP2C phosphatase. Several PP2C phosphatases belonging to clade A are involved in abscisic acid signaling and control seed dormancy. However, RDO5 does not cluster with clade A phosphatases, and abscisic acid levels and sensitivity are unaltered in the rdo5 mutant. RDO5 transcript could only be detected in seeds and was most abundant in dry seeds. RDO5 was found in cells throughout the embryo and is located in the nucleus. A transcriptome analysis revealed that several genes belonging to the conserved PUF family of RNA binding proteins, in particular Arabidopsis PUMILIO9 (APUM9) and APUM11, showed strongly enhanced transcript levels in rdo5 during seed imbibition. Further transgenic analyses indicated that APUM9 reduces seed dormancy. Interestingly, reduction of APUM transcripts by RNA interference complemented the reduced dormancy phenotype of rdo5, indicating that RDO5 functions by suppressing APUM transcript levels.
    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.

    Pectic arabinan side chains are essential for pollen cell wall integrity during pollen development
    Cankar, K. ; Kortstee, A.J. ; Toonen, M.A.J. ; Wolters-Arts, M. ; Houbein, R. ; Mariani, C. ; Ulvskov, P. ; Jorgensen, B. ; Schols, H.A. ; Visser, R.G.F. ; Trindade, L.M. - \ 2014
    Plant Biotechnology Journal 12 (2014)4. - ISSN 1467-7644 - p. 492 - 502.
    in-vivo expression - mechanical-properties - potato pectin - arabidopsis - gene - galactan - growth - biosynthesis - mutants - tubers
    Pectin is a complex polysaccharide and an integral part of the primary plant cell wall and middle lamella, contributing to cell wall mechanical strength and cell adhesion. To understand the structure–function relationships of pectin in the cell wall, a set of transgenic potato lines with altered pectin composition was analysed. The expression of genes encoding enzymes involved in pectin acetylation, degradation of the rhamnogalacturonan backbone and type and length of neutral side chains, arabinan and galactan in particular, has been altered. Upon crossing of different transgenic lines, some transgenes were not transmitted to the next generation when these lines were used as a pollen donor, suggesting male sterility. Viability of mature pollen was severely decreased in potato lines with reduced pectic arabinan, but not in lines with altered galactan side chains. Anthers and pollen of different developmental stages were microscopically examined to study the phenotype in more detail. Scanning electron microscopy of flowers showed collapsed pollen grains in mature anthers and in earlier stages cytoplasmic protrusions at the site of the of kin pore, eventually leading to bursting of the pollen grain and leaking of the cytoplasm. This phenomenon is only observed after the microspores are released and the tapetum starts to degenerate. Timing of the phenotype indicates a role for pectic arabinan side chains during remodelling of the cell wall when the pollen grain is maturing and dehydrating.
    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.
    Ex vivo transcriptional profiling reveals a common set of genes important for the adaptation of Pseudomonas aeruginosa to chronically infected host sites
    Bielecki, P. ; Komor, U. ; Bielecka, A. ; Müsken, M. ; Puchalka, J. ; Pletz, M.W. ; Ballmann, M. ; Martins Dos Santos, V.A.P. ; Weiss, S. ; Häussler, S. - \ 2013
    Environmental Microbiology 15 (2013)2. - ISSN 1462-2912 - p. 570 - 587.
    burn wound infections - biofilm formation - cystic-fibrosis - therapeutic strategies - expression - motility - mutants - protein - system - identification
    The opportunistic bacterium Pseudomonas aeruginosa is a major nosocomial pathogen causing both devastating acute and chronic persistent infections. During the course of an infection, P.¿ aeruginosa rapidly adapts to the specific conditions within the host. In the present study, we aimed at the identification of genes that are highly expressed during biofilm infections such as in chronically infected lungs of patients with cystic fibrosis (CF), burn wounds and subcutaneous mouse tumours. We found a common subset of differentially regulated genes in all three in vivo habitats and evaluated whether their inactivation impacts on the bacterial capability to form biofilms in vitro and to establish biofilm-associated infections in a murine model. Additive effects on biofilm formation and host colonization were discovered by the combined inactivation of several highly expressed genes. However, even combined inactivation was not sufficient to abolish the establishment of an infection completely. These findings can be interpreted as evidence that either redundant traits encode functions that are essential for in vivo survival and chronic biofilm infections and/or bacterial adaptation is considerably achieved independently of transcription levels. Supplemental screens, will have to be applied in order to identify the minimal set of key genes essential for the establishment of chronic infectious diseases
    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.
    My favourite flowering image
    Koornneef, M. - \ 2013
    Journal of Experimental Botany 64 (2013)18. - ISSN 0022-0957 - p. 5801 - 5803.
    arabidopsis - mutants
    I selected my favourite image from a paper by Professor Friedrich Laibach, the founder of Arabidopsis research. His paper from 1951 is the first paper dealing with natural variation for flowering time in this species, a topic many scientists including myself, have followed up and has resulted in large steps forward in our understanding of flowering time regulation. How this topic came to be of interest in my laboratory in Wageningen is described in this short overview
    ABA-deficiency results in reduced plant and fruit size in tomato
    Nitsch, L. ; Kohlen, W. ; Oplaat, C. ; Charnikhova, T. ; Cristescu, S. ; Michieli, P. ; Wolters-Arts, M. ; Bouwmeester, H.J. ; Mariani, C. ; Vriezen, W.H. ; Rieu, I. - \ 2012
    Journal of Plant Physiology 169 (2012)9. - ISSN 0176-1617 - p. 878 - 883.
    abscisic-acid biosynthesis - shoot growth - arabidopsis-thaliana - endogenous aba - ethylene - mutants - drought - stress - gene - expression
    Abscisic acid (ABA) deficient mutants, such as notabilis and flacca, have helped elucidating the role of ABA during plant development and stress responses in tomato (Solanum lycopersicum L.). However, these mutants have only moderately decreased ABA levels. Here we report on plant and fruit development in the more strongly ABA-deficient notabilis/flacca (not/flc) double mutant. We observed that plant growth, leaf-surface area, drought-induced wilting and ABA-related gene expression in the different genotypes were strongly correlated with the ABA levels and thus most strongly affected in the not/flc double mutants. These mutants also had reduced fruit size that was caused by an overall smaller cell size. Lower ABA levels in fruits did not correlate with changes in auxin levels, but were accompanied by higher ethylene evolution rates. This suggests that in a wild-type background ABA stimulates cell enlargement during tomato fruit growth via a negative effect on ethylene synthesis.
    A naturally occurring InDel variation in BraA.FLC.b(BrFLC2) associated with flowering time variation in Brassica rapa
    Wu, J. ; Wei, K.Y. ; Cheng, F. ; Li, S.K. ; Wang, Q. ; Jianjun Zhao, Jianjun ; Bonnema, A.B. ; Wang, X.W. - \ 2012
    BMC Plant Biology 12 (2012). - ISSN 1471-2229 - 9 p.
    locus-c flc - arabidopsis-thaliana - gene - vernalization - frigida - phenotype - mutants - protein
    Background: Flowering time is an important trait in Brassica rapa crops. FLOWERING LOCUS C (FLC) is a MADS-box transcription factor that acts as a potent repressor of flowering. Expression of FLC is silenced when plants are exposed to low temperature, which activates flowering. There are four copies of FLC in B. rapa. Analyses of different segregating populations have suggested that BraA.FLC.a (BrFLC1) and BraA.FLC.b (BrFLC2) play major roles in controlling flowering time in B. rapa. Results: We analyzed the BrFLC2 sequence in nine B. rapa accessions, and identified a 57-bp insertion/deletion (InDel) across exon 4 and intron 4 resulting in a non-functional allele. In total, three types of transcripts were identified for this mutated BrFLC2 allele. The InDel was used to develop a PCR-based marker, which was used to screen a collection of 159 B. rapa accessions. The deletion genotype was present only in oil-type B. rapa, including ssp. oleifera and ssp. tricolaris, and not in other subspecies. The deletion genotype was significantly correlated with variation in flowering time. In contrast, the reported splicing site variation in BrFLC1, which also leads to a non-functional locus, was detected but not correlated with variation in flowering time in oil-type B. rapa, although it was correlated with variation in flowering time in vegetable-type B. rapa. Conclusions: Our results suggest that the naturally occurring deletion mutation across exon 4 and intron 4 in BrFLC2 gene contributes greatly to variation in flowering time in oil-type B. rapa. The observed different relationship between BrFLC1 or BrFLC2 and flowering time variation indicates that the control of flowering time has evolved separately between oil-type and vegetable-type B. rapa groups.
    OSCILLATOR: A system for analysis of diurnal leaf growth using infrared photography combined with wavelet transformation
    Bours, R.M.E.H. ; Muthuraman, M. ; Bouwmeester, H.J. ; Krol, A.R. van der - \ 2012
    Plant Methods 8 (2012). - ISSN 1746-4811
    arabidopsis-thaliana - circadian clock - ethylene - plant - movement - mutants - rhythms - angle
    Background Quantification of leaf movement is an important tool for characterising the effects of environmental signals and the circadian clock on plant development. Analysis of leaf movement is currently restricted by the attachment of sensors to the plant or dependent upon visible light for time-lapse photography. The study of leaf growth movement rhythms in mature plants under biological relevant conditions, e.g. diurnal light and dark conditions, is therefore problematic. Results Here we present OSCILLATOR, an affordable system for the analysis of rhythmic leaf growth movement in mature plants. The system contains three modules: (1) Infrared time-lapse imaging of growing mature plants (2) measurement of projected distances between leaf tip and plant apex (leaf tip tracking growth-curves) and (3) extraction of phase, period and amplitude of leaf growth oscillations using wavelet analysis. A proof-of-principle is provided by characterising parameters of rhythmic leaf growth movement of different Arabidopsis thaliana accessions as well as of Petunia hybrida and Solanum lycopersicum plants under diurnal conditions. The amplitude of leaf oscillations correlated to published data on leaf angles, while amplitude and leaf length did not correlate, suggesting a distinct leaf growth profile for each accession. Arabidopsis mutant accession Landsberg erecta displayed a late phase (timing of peak oscillation) compared to other accessions and this trait appears unrelated to the ERECTA locus. Conclusions OSCILLATOR is a low cost and easy to implement system that can accurately and reproducibly quantify rhythmic growth of mature plants for different species under diurnal light/dark cycling.
    Seed maturation in Arabidopsis is characterised by nuclear size reduction and increased chromatin condensation
    Zanten, M. van; Koini, M.A. ; Geyer, R. ; Liu, Y. ; Brambilla, V. ; Bartels, D. ; Koornneef, M. ; Fransz, P. ; Soppe, W.J.J. - \ 2011
    Proceedings of the National Academy of Sciences of the United States of America 108 (2011)50. - ISSN 0027-8424 - p. 20219 - 20224.
    plant craterostigma-plantagineum - desiccation tolerance - gene-regulation - dormancy - germination - heterochromatin - mutants - establishment - transcription - organization
    Most plant species rely on seeds for their dispersal and survival under unfavorable environmental conditions. Seeds are characterized by their low moisture content and significantly reduced metabolic activities. During the maturation phase, seeds accumulate storage reserves and become desiccation-tolerant and dormant. Growth is resumed after release of dormancy and the occurrence of favorable environmental conditions. Here we show that embryonic cotyledon nuclei of Arabidopsis thaliana seeds have a significantly reduced nuclear size, which is established at the beginning of seed maturation. In addition, the chromatin of embryonic cotyledon nuclei from mature seeds is highly condensed. Nuclei regain their size and chromatin condensation level during germination. The reduction in nuclear size is controlled by the seed maturation regulator ABSCISIC ACID-INSENSITIVE 3, and the increase during germination requires two predicted nuclear matrix proteins, LITTLE NUCLEI 1 and LITTLE NUCLEI 2. Our results suggest that the specific properties of nuclei in ripe seeds are an adaptation to desiccation, independent of dormancy. We conclude that the changes in nuclear size and chromatin condensation in seeds are independent, developmentally controlled processes
    The D-galacturonic acid catabolic pathway in Botrytis cinerea
    Zhang, L. ; Thiewes, H. ; Kan, J.A.L. van - \ 2011
    Fungal Genetics and Biology 48 (2011)10. - ISSN 1087-1845 - p. 990 - 997.
    mold hypocrea-jecorina - aspergillus-nidulans - filamentous fungi - identification - pathogenesis - expression - virulence - aldolase - mutants - enzymes
    d-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving d-galacturonate reductase, l-galactonate dehydratase, and 2-keto-3-deoxy-l-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire d-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-l-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on d-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the d-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.
    Comparison of two GM maize varieties with a near-isogenic non-GM variety using transcriptomics, proteomics and metabolomics
    Barros, E. ; Lezar, S. ; Anttonen, M.J. ; Dijk, J.P. van; Rohlig, R.M. ; Kok, E.J. ; Engel, K.H. - \ 2010
    Plant Biotechnology Journal 8 (2010)4. - ISSN 1467-7644 - p. 436 - 451.
    gene-expression - h-1-nmr spectroscopy - safety assessment - food - tool - nmr - hybridization - microarrays - mutants - plants
    P>The aim of this study was to evaluate the use of four nontargeted analytical methodologies in the detection of unintended effects that could be derived during genetic manipulation of crops. Three profiling technologies were used to compare the transcriptome, proteome and metabolome of two transgenic maize lines with the respective control line. By comparing the profiles of the two transgenic lines grown in the same location over three growing seasons, we could determine the extent of environmental variation, while the comparison with the control maize line allowed the investigation of effects caused by a difference in genotype. The effect of growing conditions as an additional environmental effect was also evaluated by comparing the Bt-maize line with the control line from plants grown in three different locations in one growing season. The environment was shown to play an important effect in the protein, gene expression and metabolite levels of the maize samples tested where 5 proteins, 65 genes and 15 metabolites were found to be differentially expressed. A distinct separation between the three growing seasons was also found for all the samples grown in one location. Together, these environmental factors caused more variation in the different transcript/protein/metabolite profiles than the different genotypes.
    Involvement of the mannose phosphotransferase system of Lactobacillus plantarum WCFS1 in peroxide stress tolerance
    Stevens, M.J.A. ; Molenaar, D. ; Jong, A. de; Vos, W.M. de; Kleerebezem, M. - \ 2010
    Applied and Environmental Microbiology 76 (2010)11. - ISSN 0099-2240 - p. 3748 - 3752.
    lactic-acid bacteria - lactate utilization - oxygen - mutants - glucose
    A Lactobacillus plantarum strain with a deletion in the gene rpoN, encoding the alternative sigma factor 54 (sigma(54)), displayed a 100-fold-higher sensitivity to peroxide than its parental strain. This feature could be due to sigma(54)-dependent regulation of genes involved in the peroxide stress response. However, transcriptome analyses of the wild type and the mutant strain during peroxide exposure did not support such a role for sigma(54). Subsequent experiments revealed that the impaired expression of the mannose phosphotransferase system (PTS) operon in the rpoN mutant caused the observed increased peroxide sensitivity
    Functional study on GTP hydrolysis by the GTP-binding protein from Sulfolobus solfataricus, a member of the HflX family
    Huang, B. ; Wu, H. ; Hao, N. ; Blombach, F. ; Oost, J. van der; Li, X. ; Zhang, X.C. ; Rao, Z. - \ 2010
    Journal of Biochemistry 148 (2010)1. - ISSN 0021-924X - p. 103 - 113.
    ras p21 - activation - mutants - gtpases - loop - substitutions - purification - resolution - software - complex
    GTPase domains from members of the HflX protein family have their catalytic glutamine residue of the DxxGQ motif substituted by phenylalanine, while they are still able to hydrolyse GTP. This appears to challenge the traditional view of GTP hydrolysis mechanism of Ras-like GTPases. SsGBP from the hyperthermophilic archaeon Sulfolobus solfataricus provided the first crystal structure of the HflX family. Here, we report structure-based mutagenesis analyses on SsGBP. Six-point mutations were individually introduced in the Ras-like GTPase domain including regions of P-loop, switches I and II. Intrinsic GTPase activities and thermal stabilities of these variants together with the wild-type full-length SsGBP and its isolated GTPase domain were analysed. Both functional and structural analyses of G235P and G235S mutants, which showed total and partial loss of the GTP hydrolyzing activity, respectively, support our hypothesis that the role of aligning a nucleophilic water molecule by the Ras Gln60 residue is replaced by the backbone amide group of Gly235 in SsGBP. Together with functional studies of other mutants, we conclude that the classical view of GTP hydrolysis mechanism likely remains the same in the HflX family with a twist in the entity of the nucleophilic alignment
    A nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis
    Wang, D. ; Griffitts, J. ; Starker, C. ; Fedorova, E. ; Limpens, E.H.M. ; Ivanov, S.E. ; Bisseling, T. ; Long, S. - \ 2010
    Science 327 (2010)5969. - ISSN 0036-8075 - p. 1126 - 1129.
    signal peptidase activity - medicago-truncatula - root-nodules - endoplasmic-reticulum - gene-expression - membrane - fixation - mutants - define - plant
    The nitrogen-fixing symbiosis between Sinorhizobium meliloti and its leguminous host plant Medicago truncatula occurs in a specialized root organ called the nodule. Bacteria that are released into plant cells are surrounded by a unique plant membrane compartment termed a symbiosome. We found that in the symbiosis-defective dnf1 mutant of M. truncatula, bacteroid and symbiosome development are blocked. We identified the DNF1 gene as encoding a subunit of a signal peptidase complex that is highly expressed in nodules. By analyzing data from whole-genome expression analysis, we propose that correct symbiosome development in M. truncatula requires the orderly secretion of protein constituents through coordinated up-regulation of a nodule-specific pathway exemplified by DNF1
    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.
    Germinator: A software package for high-throughput scoring and curve fitting of Arabidopsis seed germination
    Joosen, R.V.L. ; Kodde, J. ; Willems, L.A.J. ; Ligterink, W. ; Plas, L.H.W. van der; Hilhorst, H.W.M. - \ 2010
    The Plant Journal 62 (2010)1. - ISSN 0960-7412 - p. 148 - 159.
    quantitative trait loci - inbred line population - image-analysis - thaliana - tolerance - dormancy - mutants - stress - biosynthesis - expression
    Over the past few decades seed physiology research has contributed to many important scientific discoveries and has provided valuable tools for the production of high quality seeds. An important instrument for this type of research is the accurate quantification of germination; however gathering cumulative germination data is a very laborious task which is often prohibitive to the execution of large experiments. In this paper we present the Germinator package: a simple, highly cost efficient and flexible procedure for high-throughput automatic scoring and evaluation of germination that can be implemented without the use of complex robotics. The Germinator package contains three modules; 1) design of experimental setup with various options to replicate and randomize samples; 2) automatic scoring of germination based on the color contrast between the protruding radicle and seed coat on a single image; 3) curve fitting of cumulative germination data and the extraction, recap and visualization of the various germination parameters. The curve fitting module enables analysis of general cumulative germination data and can be used for all plant species. We show that the automatic scoring system works for Arabidopsis thaliana and Brassica spp. seeds, but is likely to be applicable to other species, as well. In this paper we show the accuracy, reproducibility and flexibility of the Germinator package. We have successfully applied it to evaluate natural variation for salt tolerance in a large population of Recombinant Inbred Lines (RIL) and were able to identify several QTL for salt tolerance. Germinator is a low-cost package that allows the monitoring of several thousands of germination tests, several times a day by a single person.
    Genome-wide screen for Listeria monocytogenes genes important for growth at high temperatures
    Veen, S. van der; Abee, T. ; Vos, W.M. de; Wells-Bennik, M.H.J. - \ 2009
    FEMS Microbiology Letters 295 (2009)2. - ISSN 0378-1097 - p. 195 - 203.
    staphylococcus-aureus - bacillus-subtilis - stress tolerance - escherichia-coli - cell-division - heat-shock - expression - virulence - identification - mutants
    Listeria monocytogenes is a Gram-positive food-borne pathogen that is able to grow over a wide temperature range. Although the class I and class III heat-shock genes are known to play an important role in heat shock, information on genes that are essential for growth at high temperatures is scarce. To determine which genes are important for growth at high temperatures (42.5-43 degrees C), we performed a random insertion screening in L. monocytogenes, rendering 28 temperature-sensitive mutants. These mutants showed insertions in genes that play a role in transcription regulation, cell-wall biosynthesis, cell division, translation, transport, sensing, and specific stress responses like the SOS response and the class III heat-shock response. Some of these mutants showed altered morphological characteristics such as cell elongation, reduced cell length, or sickle shapes. Furthermore, the majority of the mutants showed increased heat inactivation after exposure to 55 degrees C compared with the wild-type strain. The role of the specific genes in relation to growth at high temperatures is discussed
    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
    Genes involved in carotene synthesis and mating in Blakeslea trispora
    Kuzina, V. ; Ramirez-Medina, H. ; Visser, H. ; Ooyen, A.J.J. van; Cerda-Olmedo, E. ; Berg, J.A. van den - \ 2008
    Current Genetics 54 (2008)3. - ISSN 0172-8083 - p. 143 - 152.
    beta-carotene - cdna-aflp - saccharomyces-cerevisiae - expression data - biosynthesis - phycomyces - light - mucorales - lycopene - mutants
    Mating of Blakeslea trispora and other molds of the order Mucorales requires the interaction of mycelia of opposite sex, (+) and (-), leading to the development of specialized structures and to an enhanced accumulation of beta-carotene. Industry obtains beta-carotene by co-cultivating appropriate strains of Blakeslea (mated cultures). Gene transcription in single and mated cultures was assayed by cDNA-AFLP, a technique to observe the differential expression of subsets of mRNA fragments. Overexpression in mated cultures is about ten times more frequent than underexpression. We obtained and sequenced fragments of 97 candidate genes that appeared to be overexpressed during mating and confirmed four of them by reverse transcription and real-time PCR. Comparisons with gene sequences from other organisms suggest functions in carotene biosynthesis (4 genes), energy metabolism (8), cell wall synthesis (1), transfer of acetyl groups (1), and regulatory processes (10). Sodium acetate inhibited sexual overexpression in about two-thirds of the candidate genes and acted as a signal with broad effects on the metabolism and the morphology of mated cultures. Our work offers new materials for the study of carotene biosynthesis and its regulation and for the improvement of carotene production with Mucorales.
    Development of a quantitative real-time PCR for determination of genotype frequencies for studies in baculovirus population biology
    Zwart, M.P. ; Oers, M.M. van; Cory, J.S. ; Lent, J.W.M. van; Werf, W. van der; Vlak, J.M. - \ 2008
    Journal of Virological Methods 148 (2008)1-2. - ISSN 0166-0934 - p. 146 - 154.
    nuclear polyhedrosis-virus - polymerase-chain-reaction - trichoplusia-ni - sybr-green - mosaic-virus - wild-type - rt-pcr - nucleopolyhedrovirus - mutants - larvae
    Two bacmid-derived Autographa californica Multiple-capsid Nucleopolyhedrovirus genotypes ¿ that differ only in a short tag sequence for differential PCR recognition ¿ were generated. By electron microscopy, these genotypes were found to have identical polyhedra morphology. Mixtures of quantified polyhedra were made and used to validate a SYBR Green I-based quantitative real-time PCR (qPCR) to determine genotype frequencies in mixed genotype populations. The PCR could accurately quantify genotype ratios over a range of 8 orders of magnitude. Only a small correction of the genotype ratio was necessary to obtain a valid result. Low levels of aspecific background (a fluorescent signal when the template corresponding with the primer set used is not present) were measured in these validation experiments and in a typical laboratory setup. A small fitness difference between the genotypes generated was observed in a median lethal dose bioassay. The bacmid-derived virus genotypes generated and the qPCR assays are valuable tools for studying the population biology of baculoviruses.
    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.






    Global analysis of gene expression in flower buds of Ms-cd1 Brassica oleracea conferring male sterility by using an Arabidopsis microarray
    Kang, Jungen ; Zhang, Guoyu ; Bonnema, A.B. ; Fang, Zhiyuan ; Wang, Xiaowu - \ 2008
    Plant Molecular Biology 66 (2008)1-2. - ISSN 0167-4412 - p. 177 - 192.
    male gametophyte development - lathyrus-odoratus l - pollen-tube growth - mother cell-wall - pectin methylesterase - anther development - thaliana - identification - mutants - transcriptome
    The dominant male sterility gene Ms-cd1 is identified in Brassica oleracea. Electron microscopical observations revealed that abortion of pollen development starts after tetrad formation. This important male sterility phenotype is characterized by lack of degradation of the primary pollen mother cell (PMC) wall and delayed degradation of callose surrounding the tetrads and thus arrest of microspore release. Gene expression of the male sterile and fertile buds was analyzed by heterologous hybridization of Brassica oleracea cRNA onto an Arabidopsis whole genome oligonucleotide microarray. A total of 277 suppressed genes including 40 kinase-, 32 cell wall modification and 29 transport related genes were found to be significantly down regulated >3-fold in the male sterile mutant. The vast majority of the differentially expressed transcripts are found to present late pollen stage specific genes. Kinase genes, cell wall modification genes and ion transport genes were greatly over-represented when compared to their percentage of all flower bud expressed genes and represent 36.5% of the genes suppressed by Ms-cd1. Our results also suggest that Ms-cd1 may blocks an anther developmental pathway with a small number of genes suppressed in tapetum cells which prevent the degradation of callose and PMC wall, which further leads to the suppression of a large number of genes involved in signaling pathways, cell wall modification and ion transport in pollen grains.
    Afweermutanten
    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
    Tagging target genes of the mat1-2-1 transcription factor in Fusarium verticillioides (Gibberella fujikuroi MP-A)
    Keszthelyi, A. ; Jeney, A. ; Kerenyi, Z. ; Mendes, O. ; Waalwijk, C. ; Hornok, L. - \ 2007
    Antonie van Leeuwenhoek: : Nederlandsch tijdschrift voor hygiëne, microbiologie en serologie 91 (2007)4. - ISSN 0003-6072 - p. 373 - 391.
    pheromone precursor genes - mating-type gene - neurospora-crassa - sordaria-macrospora - sexual development - species complex - regulated genes - mutants - zeae - complementation
    Mating type in filamentous ascomycetes is controlled by idiomorphic alleles, named MAT1-1 and MAT1-2, which contain 1-3 genes. Of these genes MAT1-1-1 and MAT1-2-1 encode putative transcription factors and are thus considered to be the major regulators of sexual communication and mating. Fungi with no known sexual stage may also have fully functional mating type genes and therefore it was plausible to hypothesize that the MAT products may also regulate other types of genes not involved directly in the mating process. To identify putative target genes of these transcription factors in Fusarium verticillioides, ¿MAT1-2-1 knock out mutants were produced and transcript profiles of mutant and wild type were compared by means of differential cDNA hybridization. Clones, either up- or down-regulated in the ¿MAT1-2-1 mutant were sequenced and a total of 248 sequences were blasted against the NCBI database as well as the Gibberella zeae and Gibberella moniliformis genomes. Fifty-five percent of the clones were down-regulated in the mutant, indicating that the MAT1-2-1 product positively affected these tagged sequences. On the other hand, 45% were found to be up-regulated in the mutant, suggesting that the MAT1-2-1 product also exerted a negative regulatory function on this set of genes. Sequences involved in protein synthesis and metabolism occurred more frequently among the clones up-regulated in the mutant, whereas genes belonging to cell signalling and communication were especially frequently tagged among the sequences down-regulated in the mutant
    Medicago LYK3, an entry receptor in rhizobial nodulation factor signaling
    Smit, P. ; Limpens, E.H.M. ; Geurts, R. ; Fedorova, E. ; Dolgikh, E. ; Gough, C. ; Bisseling, T. - \ 2007
    Plant Physiology 145 (2007). - ISSN 0032-0889 - p. 183 - 191.
    protein-kinase - lotus-japonicus - calcium spiking - truncatula - mutants - gene - domain - perception - infection - responses
    Rhizobia secrete nodulation (Nod) factors, which set in motion the formation of nitrogen-fixing root nodules on legume host plants. Nod factors induce several cellular responses in root hair cells within minutes, but also are essential for the formation of infection threads by which rhizobia enter the root. Based on studies using bacterial mutants, a two-receptor model was proposed, a signaling receptor that induces early responses with low requirements toward Nod factor structure and an entry receptor that controls infection with more stringent demands. Recently, putative Nod factor receptors were shown to be LysM domain receptor kinases. However, mutants in these receptors, in both Lotus japonicus (nfr1 and nfr5) and Medicago truncatula (Medicago; nfp), do not support the two-receptor model because they lack all Nod factor-induced responses. LYK3, the putative Medicago ortholog of NFR1, has only been studied by RNA interference, showing a role in infection thread formation. Medicago hair curling (hcl) mutants are unable to form curled root hairs, a step preceding infection thread formation. We identified the weak hcl-4 allele that is blocked during infection thread growth. We show that HCL encodes LYK3 and, thus, that this receptor, besides infection, also controls root hair curling. By using rhizobial mutants, we also show that HCL controls infection thread formation in a Nod factor structure-dependent manner. Therefore, LYK3 functions as the proposed entry receptor, specifically controlling infection. Finally, we show that LYK3, which regulates a subset of Nod factor-induced genes, is not required for the induction of NODULE INCEPTION.
    An evolvable oestrogen receptor activity sensor: development of a modular system for integrating multiple genes into the yeast genome
    Fox, J.E. ; Bridgham, J.T. ; Bovee, T.F.H. ; Thornton, J.W. - \ 2007
    Yeast 24 (2007)5. - ISSN 0749-503X - p. 379 - 390.
    green fluorescent protein - saccharomyces-cerevisiae - antiestrogenic activities - directed evolution - in-vitro - populations - adaptation - efficient - mutants - assay
    To study a gene interaction network, we developed a gene-targeting strategy that allows efficient and stable genomic integration of multiple genetic constructs at distinct target loci in the yeast genome. This gene-targeting strategy uses a modular plasmid with a recyclable selectable marker and a multiple cloning site into which the gene of interest is cloned, flanked by two long regions of homology to the target genomic locus that are generated using adaptamer primers. We used this strategy to integrate into a single yeast strain components of the oestrogen receptor (ER) signalling network, comprising the human ER and three reporter genes driven by oestrogen response elements (EREs). The engineered strain contains multiple reporters of ligand-dependent receptor signalling, providing sensitive, reproducible, rapid, low-cost quantitative assays of ER activity in order to screen potential receptor agonists. Further, because two of the ERE-driven reporter genes are required for growth in deficient media, the strain's growth rate - and therefore its fitness - depends on ligand-induced ER activity. This evolvable oestrogen receptor activity sensor (EERAS) can therefore provide the foundation of a long-term experimental evolution strategy to elucidate ER structure-function relations and ligand-receptor evolution.
    Control of FWA gene silencing in Arabidopsis thaliana by SINE-related direct repeats
    Kinoshita, Y. ; Saze, H. ; Kinoshita, T. ; Miura, A. ; Soppe, W.J. ; Koornneef, M. ; Kakutani, T. - \ 2007
    The Plant Journal 49 (2007)1. - ISSN 0960-7412 - p. 38 - 45.
    novo dna methylation - medea polycomb gene - epigenetic control - cytosine methylation - tandem repeats - small rnas - transposons - maintenance - mutants - locus
    A unique feature of late-flowering fwa epigenetic mutations is that the phenotype is caused by ectopic expression of the homeobox gene FWA. During normal development the FWA gene is expressed specifically in the endosperm in an imprinted manner. Ectopic FWA expression and disruption of imprinting can be induced in mutants of a CG methyltransferase MET1 (methyltransferase 1) or a chromatin-remodeling gene DDM1 (decrease in DNA methylation 1), suggesting that the proper FWA expression depends on cytosine methylation. However, critical methylated residues controlling FWA silencing are not pinpointed. Nor is it understood how the FWA gene is initially methylated and silenced in wild-type plants. Here we mapped sequences critical for FWA silencing by application of RdDM (RNA-directed DNA methylation) to a ddm1-induced stable fwa epiallele. Transcription of double-stranded RNA corresponding to the tandem direct repeats around the FWA transcription start site induced de novo DNA methylation, transcriptional suppression and phenotypic reversion. The induced changes were heritable even without the transgene, which correlates with inheritance of CG methylation in the direct repeats. The newly silenced FWA allele was transcribed in an endosperm-specific and imprinted manner, as is the case for the wild-type FWA gene. The results indicate that methylation of the direct repeats, which presumably originated from a short interspersed nuclear element (SINE), is sufficient to induce proper epigenetic control of the FWA gene
    New Arabidopsis recombinant inbred line populations genotyped using SNPWave and their use for mapping flowering-time quantitative trait loci.
    El-Lithy, M.E.M. ; Bentsink, L. ; Hanhart, C.J. ; Ruys, G.J. ; Rovito, D. ; Broekhof, J.L.M. ; Poel, H.J. van der; Eijk, M.J. ; Vreugdenhil, D. ; Koornneef, M. - \ 2006
    Genetics 172 (2006)3. - ISSN 0016-6731 - p. 1867 - 1876.
    natural allelic variation - landsberg erecta - qtl analysis - thaliana - accessions - polymorphism - expression - ecotypes - mutants - markers
    The SNPWave marker system, based on SNPs between the reference accessions Colombia-0 and Landsberg erecta (Ler), was used to distinguish a set of 92 Arabidopsis accessions from various parts of the world. In addition, we used these markers to genotype three new recombinant inbred line populations for Arabidopsis, having Ler as a common parent that was crossed with the accessions Antwerp-1, Kashmir-2, and Kondara. The benefit of using multiple populations that contain many similar markers and the fact that all markers are linked to the physical map of Arabidopsis facilitates the quantitative comparison of maps. Flowering-time variation was analyzed in the three recombinant inbred line populations. Per population, four to eight quantitative trait loci (QTL) were detected. The comparison of the QTL positions related to the physical map allowed the estimate of 12 different QTL segregating for flowering time for which Ler has an allele different from one, two, or three of the other accessions
    Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis
    Bentsink, L. ; Jowett, J. ; Hanhart, C.J. ; Koornneef, M. - \ 2006
    Proceedings of the National Academy of Sciences of the United States of America 103 (2006)45. - ISSN 0027-8424 - p. 17042 - 17047.
    natural allelic variation - thaliana l heynh - abscisic-acid - gibberellin biosynthesis - embryo development - germination - mutants - gene - longevity - maintenance
    Genetic variation for seed dormancy in nature is a typical quantitative trait controlled by multiple loci on which environmental factors have a strong effect. Finding the genes underlying dormancy quantitative trait loci is a major scientific challenge, which also has relevance for agriculture and ecology. In this study we describe the identification of the DELAY OF GERMINATION 1 (DOG1) gene previously identified as a quantitative trait locus involved in the control of seed dormancy. This gene was isolated by a combination of positional cloning and mutant analysis and is absolutely required for the induction of seed dormancy. DOG1 is a member of a small gene family of unknown molecular function, with five members in Arabidopsis. The functional natural allelic variation present in Arabidopsis is caused by polymorphisms in the cis-regulatory region of the DOG1 gene and results in considerable expression differences between the DOG1 alleles of the accessions analyzed
    Exogenous gibberellins inhibit coffee (Coffea arabica cv. Rubi) seed germination and cause cell death in the embryo
    Silva, E.A.A. Da; Toorop, P.E. ; Nijsse, J. ; Bewley, J.D. ; Hilhorst, H.W.M. - \ 2005
    Journal of Experimental Botany 56 (2005)413. - ISSN 0022-0957 - p. 1029 - 1038.
    abscisic-acid - endosperm - arabidopsis - mutants - tomato - elongation - metabolism - induction - viability - aleurone
    The mechanism of inhibition of coffee (Coffea arabica cv. Rubi) seed germination by exogenous gibberellins (GAs) and the requirement of germination for endogenous GA were studied. Exogenous GA4+7 inhibited coffee seed germination. The response to GA4+7 showed two sensitivity thresholds: a lower one between 0 and 1 µM and a higher one between 10 and 100 µM. However, radicle protrusion in coffee seed depended on the de novo synthesis of GAs. Endogenous GAs were required for embryo cell elongation and endosperm cap weakening. Incubation of coffee seed in exogenous GA4+7 led to loss of embryo viability and dead cells were observed by low temperature scanning microscopy only when the endosperm was surrounding the embryo. The results described here indicate that the inhibition of germination by exogenous GAs is caused by factors that are released from the endosperm during or after its weakening, causing cell death in the embryo and leading to inhibition of radicle protrusion.
    Differentially expressed genes associated with dormancy or germination of Arabidopsis thaliana seeds
    Toorop, P.E. ; Barroco, R.M. ; Engler, G. ; Groot, S.P.C. ; Hilhorst, H.W.M. - \ 2005
    Planta 221 (2005)5. - ISSN 0032-0935 - p. 637 - 647.
    zaadkieming - genen - genexpressie - seed germination - genes - gene expression - ribosomal-protein genes - low-temperature - abscisic-acid - l heynh - gibberellins - desiccation - pathways - mutants - bodies - light
    Differential display analysis using dormant and non-dormant Arabidopsis thaliana (L.) Heynh seeds resulted in a set of genes that were associated with either dormancy or germination. Expression of the germination-associated genes AtRPL36B and AtRPL27B, encoding two ribosomal proteins, was undetectable in the dry seed, low in dormant seed, and high under conditions that allowed completion of germination. Expression of these genes was also found to be light-regulated and to correlate with germination speed. Expression of the dormancy-associated genes ATS2 and ATS4, encoding a caleosin-like protein and a protein similar to a low-temperature-induced protein respectively, was high in the dry seed and decreased during germination. Expression of ATS2 and ATS4 was high in primary and secondary dormant seed but low in after-ripened or chilled seed. The expression of both genes was also light-regulated, but no relationship with temperature-dependent germination speed was found.
    Ethanol breaks dormancy of the potato tuber apical bud
    Claassens, M.M.J. ; Verhees, J.A. ; Plas, L.H.W. van der; Krol, A.R. van der; Vreugdenhil, D. - \ 2005
    Journal of Experimental Botany 56 (2005)419. - ISSN 0022-0957 - p. 2515 - 2525.
    one-leaf cuttings - solanum-tuberosum - abscisic-acid - 2nd growth - germination - expression - gibberellins - arabidopsis - gene - mutants
    Growing potato tubers or freshly harvested mature tubers have a dormant apical bud. Normally, this dormancy is spontaneously broken after a period of maturation of the tuber, resulting in the growth of a new sprout. Here it is shown that in in vitro-cultured growing and maturing tubers, ethanol can rapidly break this dormancy and re-induce growth of the apical bud. The in vivo promoter activity of selected genes during this secondary growth of the apical bud was monitored, using luciferase as a reporter. In response to ethanol, the expression of carbohydrate-storage, protein-storage, and cell division-related genes are rapidly down-regulated in tuber tissue. It was shown that dormancy was broken by primary but not by secondary alcohols, and the effect of ethanol on sprouting and gene expression in tuber tissue was blocked by an inhibitor of alcohol dehydrogenase. By contrast, products derived from alcohol dehydrogenase activity (acetaldehyde and acetic acid) did not induce sprouting, nor did they affect luciferase reporter gene activity in the tuber tissue. Application of an inhibitor of gibberellin biosynthesis had no effect on ethanol-induced sprouting. It is suggested that ethanol-induced sprouting may be related to an alcohol dehydrogenase-mediated increase in the catabolic redox charge [NADH/(NADH+NAD+)].
    NSP1 of the GRAS protein family is essential for Rhizobial Nod factor-induced transcription
    Smit, P. ; Raedts, J.G.J. ; Portyanko, V. ; Debellé, F. ; Gough, C. ; Bisseling, T. ; Geurts, R. - \ 2005
    Science 308 (2005)5729. - ISSN 0036-8075 - p. 1789 - 1791.
    receptor kinase gene - medicago-truncatula - bacterial - fungal - identification - transduction - symbiosis - mutants - calcium
    Rhizobial Nod factors induce in their legume hosts the expression of many genes and set in motion developmental processes leading to root nodule formation. Here we report the identification of the Medicago GRAS-type protein Nodulation signaling pathway 1 (NSP1), which is essential for all known Nod factor–induced changes in gene expression. NSP1 is constitutively expressed, and so it acts as a primary transcriptional regulator mediating all known Nod factor–induced transcriptional responses, and therefore, we named it a Nod factor response factor.
    Probing solvent accessibility of transthyretin amyloid by solution NMR spectroscopy
    Olofsson, A. ; Ippel, J.H. ; Wijmenga, S.S. ; Lundgren, E. ; Ohman, A. - \ 2004
    Journal of Biological Chemistry 279 (2004)7. - ISSN 0021-9258 - p. 5699 - 5707.
    hydrogen-exchange - tetramer dissociation - subunit interface - x-ray - fibrils - core - intermediate - proteins - variants - mutants
    The human plasma protein transthyretin (TTR) may form fibrillar protein deposits that are associated with both inherited and idiopathic amyloidosis. The present study utilizes solution nuclear magnetic resonance spectroscopy, in combination with hydrogen/deuterium exchange, to determine residue-specific solvent protection factors within the fibrillar structure of the clinically relevant variant, TTRY114C. This novel approach suggests a fibril core comprised of the six beta-strands, A-B-E-F-G-H, which retains a native-like conformation. Strands C and D are dislocated from their native edge region and become solvent-exposed, leaving a new interface involving strands A and B open for intermolecular interactions. Our results further support a native-like intermolecular association between strands F-F' and H-H' with a prolongation of these beta-strands and, interestingly, with a possible shift in beta-strand register of the subunit assembly. This finding may explain previous observations of a monomeric intermediate preceding fibril formation. A structural model based on our results is presented.
    AtGA3ox2, a key gene responsible for bioactive gibberellin biosynthesis, is regulated during embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis
    Curaba, J. ; Moritz, T. ; Blervaque, R. ; Parcy, F. ; Raz, V. ; Herzog, M. ; Vachon, G. - \ 2004
    Plant Physiology 136 (2004)3. - ISSN 0032-0889 - p. 3660 - 3669.
    late embryo development - thaliana l heynh - abscisic-acid - seed development - leafy cotyledon1 - transcription factor - fus3 gene - mutants - germination - expression
    Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both B-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.
    In vitro characterization of ge negative bovine herpesvirus types 1.1 (BHV-1.1) and 1.2a (BHV-1.2a)
    Spilki, F.R. ; Franco, A.C. ; Rijsewijk, F.A.M. ; Weiblen, R. ; Flores, E.F. ; Roehe, P.M. - \ 2004
    Brazilian Journal of Microbiology 35 (2004)3. - ISSN 1517-8382 - p. 264 - 268.
    to-cell spread - glycoprotein-e - simplex-virus - gi - junctions - deletion - mutants - gg
    This study aimed the in vitro growth characterization of a previously constructed Brazilian bovine herpesvirus 1.2a with a deletion in the glycoprotein E gene (BHV-1.2a gE-). The plaque sizes, penetration and growth kinetics of the Brazilian BHV-1.2a gE- were studied and compared with the parental virus, as well as with a BHV-1.1 gE- recombinant derived from an European BHV-1.1 strain. No statistical differences were observed between the gE- recombinants and the respective parental viruses penetration assays were performed. When single step growth curves were studied, no statistical differences were observed between gE- and parental viruses. However, it was observed that both gE- viruses were excreted from cells in significantly higher titres at 11 hours post infection in comparison with parental viruses. No statistical differences were observed when plaque sizes of parental viruses or gE- viruses we analyzed separately in each cell type. However, both gE- recombinants displayed a significantly reduced plaque areas on three different cell cultures, in comparison with parental viruses, indicating that the lack of gE had the same effect on both BHV-1 subtypes, manifested by a restricted cell-to-cell spread in infected cells.
    Occurrence of SDR 2N-gametes in Lilium hybrids
    Lim, K.B. ; Shen, T.M. ; Barba Gonzalez, R. ; Ramanna, M.S. ; Tuyl, J.M. van - \ 2004
    Breeding Science 54 (2004)1. - ISSN 1344-7610 - p. 13 - 18.
    interspecific hybrids - bc2 progenies - potato - mutants - desynapsis - fertility - crosses - pollen - gish
    The mechanism of SDR 2n-pollen formation was analyzed in two intra-sectional diploid (2n = 2x = 24) Lilium hybrids (Enchantment x L. pumilum). Variable frequencies of 2n-pollen were found. Meiotic analysis indicated that the intra-sectional hybrids showed perfect chromosome pairing in most cases at metaphase I and normal anaphase I movement of pollen mother cells (PMCs), but produced 2n-pollen by second division restitution (SDR). A high bivalent formation (11.9II and 11.8II, respectively) at metaphase 1, irregular meiotic division such as unbalanced chromosome separation and chromatic fragmentation resulted yet in acceptable pollen fertility for cross-pollination. The hybrids were fertile, and when used as male parents, offspring could tie generated. The significance of the occurrence of 2n-pollen for the breeding of lilies was analyzed.
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