Staff Publications

Staff Publications

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

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

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

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

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    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.
    Both ApxI and ApxII of Actinobacillus pleuropneumoniae serotype 1 are necessary for full virulence
    Boekema, B.K.H.L. ; Kamp, E.M. ; Smits, M.A. ; Smith, H.E. ; Stockhofe-Zurwieden, N. - \ 2004
    Veterinary Microbiology 100 (2004)1-2. - ISSN 0378-1135 - p. 17 - 23.
    rtx-toxins - endobronchial inoculation - pigs - hemolysins - cytolysins - mutants - cells
    Most serotypes of A. pleuropneumoniae produce more than one toxin in vivo. To determine the value of the production of more than one toxin in the development of disease, we tested the pathogenicity of isogenic strains of A. pleuropneumoniae serotype 1 that are mutated in the toxin genes apxIA and/or apxIIA or in the transport genes apxIBD. Bacteria mutated in both apxIA and apxIIA, or in apxIBD, were unable to induce pathological lesions, thereby confirming the conclusion that ApxI and ApxII are essential for the pathogenesis of pleuropneumonia. Infection with isogenic strains lacking either ApxI or ApxII did not consistently lead to pleuropneumonia unlike the parent strain S4074. ApxII seemed at least as important as ApxI for the development of clinical and pathological symptoms. Only one of the four pigs inoculated with a mutant strain unable to produce ApxII developed mild pneumonia whereas two out of the three pigs inoculated with a mutant strain unable to produce ApxI developed more severe lesions. The results indicate that both ApxI and ApxII of A. pleuropneumoniae serotype 1 are necessary for full virulence.
    Mating type sequences in asexually reproducing Fusarium species
    Kenényi, Z. ; Moretti, A. ; Waalwijk, C. ; Oláh, B. ; Hornok, L. - \ 2004
    Applied and Environmental Microbiology 70 (2004)8. - ISSN 0099-2240 - p. 4419 - 4423.
    gibberella-fujikuroi - vegetative compatibility - complex - genes - amplification - oxysporum - chemistry - genetics - biology - mutants
    To assess the potential for mating in several Fusarium species with no known sexual stage, we developed degenerate and semidegenerate oligonucleotide primers to identify conserved mating type (MAT) sequences in these fungi. The putative and high-mobility-group (HMG) box sequences from Fusarium avenaceum, F. culmorum, F. poae, and F. semitectum were compared to similar sequences that were described previously for other members of the genus. The DNA sequences of the regions flanking the amplified MAT regions were obtained by inverse PCR. These data were used to develop diagnostic primers suitable for the clear amplification of conserved mating type sequences from any member of the genus Fusarium. By using these diagnostic primers, we identified mating types of 122 strains belonging to 22 species of Fusarium. The box and the HMG box from the mating type genes are transcribed in F. avenaceum, F. culmorum, F. poae, and F. semitectum. The novelty of the PCR-based mating type identification system that we developed is that this method can be used on a wide range of Fusarium species, which have proven or expected teleomorphs in different ascomycetous genera, including Calonectria, Gibberella, and Nectria
    Salmonella gene rma (ramA) and multiple-drug-resistant Salmonella enterica serovar typhimurium
    Straaten, T. van; Janssen, R. ; Mevius, D.J. ; Dissel, J.T. van - \ 2004
    Antimicrobial Agents and Chemotherapy 48 (2004)6. - ISSN 0066-4804 - p. 2292 - 2294.
    escherichia-coli - antibiotic-resistance - virulence - confers - mutants
    MarA and its homologue, RamA, have been implicated in multidrug resistance (MDR). RamA overexpression in Salmonella enterica serovar Typhimurium and Escherichia coli conferred MDR independently of marA. Inactivation of ramA did not affect the antibiotic susceptibilities of wild-type S. enterica serovar Typhimurium or 15 unrelated clinical MDR isolates. Thus, ramA overexpression is not a common MDR mechanism in Salmonella
    Molecular analysis of plant architecture in Arabidopsis thaliana using activation tagging.
    Chalfun Junior, A. - \ 2004
    Wageningen University. Promotor(en): Maarten Koornneef; Gerco Angenent. - Wageningen : S.n. - ISBN 9789085040361 - 144
    arabidopsis thaliana - transposons - dna - mutanten - plantenontwikkeling - plantenmorfologie - moleculaire genetica - genexpressie - arabidopsis thaliana - transposable elements - dna - mutants - plant development - plant morphology - molecular genetics - gene expression
    Keywords: Arabidopsisthaliana, activation tagging, T-DNA, transposon, mutants, enhancer, DNA methylation, plant architecture, development, forward/reverse genetics, lateral organs, flower, vascular tissue, HLH, transmembrane, transcription factors

    Plant development is one of the most important aspects of plant's life cycle that has extensively been studied at the morphological, genetic and molecular level. It is import for systematic and taxonomic classification, but also for applied agronomic reasons, because it affects the growth and cultivation leading to higher yield and quality of the product.The generation of genetic variants, like mutants may increase genetic pool and gives information about plant processes and their genetic control.Activation tagging is a new powerful tool to generate and identify new mutants, which emerged as an alternative for gene function analysis. This thesis reports the study on the molecular control of plant architecture, using mutants generated by an activation tagging-based approach in the model plant Arabidopsis thaliana . In addition, it also describes experiments that could explain why the low frequencies of mutants were obtained by T-DNA based activation tagging. Based on this comparison, the transposon-based activation tagging strategy was chosen and a screen for flower and silique mutants in a large Arabidopsis population yielded three gain-of-function mutants. These mutants were designated downwards siliques1 ( ds1-D ), needle1 ( ndl1-D ) and twisted1 ( twt1-D ). In the ds1-D mutant, internodes are shorter and the lateral organs such as flowers are bending downwards. Further molecular and genetic studies on this mutant revealed that DS1 is important to control petiole-blade boundary in Arabidopsis petals. In the ndl1-D mutant, the normal formation of valve tissues is altered, resulting in a pin-like structure that replaces the two fused carpels of the wild type pistil. The results suggest that NDL1 is involved in normal carpel development, in which auxin distribution plays an important role. In the third mutant, twt1-D , the overexpression of TWT1 led to twisting of all organs, whichismost pronounced in siliques. This phenotype and the expression pattern of the gene suggest that TWT1 is involved in proper vascular tissue development in Arabidopsis . These studies demonstrate the power of activation tagging and it gains valuable knowledge about the molecular networks that control plant development.
    The genetics of seed quality in Arabidopsis thaliana
    Clerkx, E.J.M. - \ 2004
    Wageningen University. Promotor(en): Maarten Koornneef, co-promotor(en): Steven Groot. - Wageningen : S.n. - ISBN 9789058089700 - 136
    zaadkwaliteit - genetische regulatie - zaadlevensduur - droogteresistentie - genetische variatie - genen - mutanten - seed quality - genetic regulation - seed longevity - drought resistance - genetic variation - genes - mutants
    Effect of adaptation to ethanol on cytoplasmic and membrane protein profiles of Oenococcus oeni
    Silveira, M.G. da; Baumgärtner, M. ; Rombouts, F.M. ; Abee, T. - \ 2004
    Applied and Environmental Microbiology 70 (2004)5. - ISSN 0099-2240 - p. 2748 - 2755.
    heat-shock-protein - mannose phosphotransferase system - carbon catabolite repression - lactic-acid bacteria - leuconostoc-oenos - lactobacillus-pentosus - multiple stresses - cells - phosphoenolpyruvate - mutants
    The practical application of commercial malolactic starter cultures of Oenococcus oeni surviving direct inoculation in wine requires insight into mechanisms of ethanol toxicity and of acquired ethanol tolerance in this organism. Therefore, the site-specific location of proteins involved in ethanol adaptation, including cytoplasmic, membrane-associated, and integral membrane proteins, was investigated. Ethanol triggers alterations in protein patterns of O. oeni cells stressed with 12% ethanol for 1 h and those of cells grown in the presence of 8% ethanol. Levels of inosine-5'-monophosphate dehydrogenase and phosphogluconate dehydrogenase, which generate reduced nicotinamide nucleotides, were decreased during growth in the presence of ethanol, while glutathione reductase, which consumes NADPH, was induced, suggesting that maintenance of the redox balance plays an important role in ethanol adaptation. Phosphoenolpyruvate:mannose phosphotransferase system (PTS) components of mannose PTS, including the phosphocarrier protein HPr and EIIMan, were lacking in ethanol-adapted cells, providing strong evidence that mannose PTS is absent in ethanol-adapted cells, and this represents a metabolic advantage to O. oeni cells during malolactic fermentation. In cells grown in the presence of ethanol, a large increase in the number of membrane-associated proteins was observed. Interestingly, two of these proteins, dTDT-glucose-4,6-dehydratase and D-alanine:D-alanine ligase, are known to be involved in cell wall biosynthesis. Using a proteomic approach, we provide evidence for an active ethanol adaptation response of O. oeni at the cytoplasmic and membrane protein levels.
    A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa
    Choi, H.K. ; Kim, D. ; Uhm, T. ; Limpens, E.H.M. ; Lim, H. ; Mun, J.H. ; Kalo, P. ; Penmetsa, R.V. ; Seres, A. ; Kulikova, O. ; Roe, B.A. ; Bisseling, T. ; Kiss, G.B. ; Cook, D.R. - \ 2004
    Genetics 166 (2004)3. - ISSN 0016-6731 - p. 1463 - 1502.
    nod factor transduction - pcr-based markers - arabidopsis-thaliana - linkage map - expression - construction - leguminosae - mutants - model - identification
    A core genetic map of the legume Medicago truncatula has been established by analyzing the segregation of 288 sequence-characterized genetic markers in an E, population composed of 93 individuals. These molecular markers correspond to 141 ESTs, 80 BAC end sequence tags, and 67 resistance gene analogs, covering 513 cM. In the case of EST-based markers we used an intron-targeted marker strategy with primers designed to anneal in conserved exon regions and to amplify across intron regions. Polymorphisms were significantly more frequent in intron vs. exon regions, thus providing an efficient mechanism to map) transcribed genes. Genetic and cytogenetic analysis produced eight well-resolved linkage groups, which have been previously correlated with eight chromosomes by means of FISH with mapped BAC clones. We anticipated that mapping of conserved coding regions would have utility for comparative mapping among legumes: thus 60 of the EST-based printer pairs were designed to amplify orthologons sequences across a range of legume species. As an initial test of this strategy, we used primers designed against M. truncatula exon sequences to rapidly map genes in M. sativa. The resulting comparative map, which includes 68 bridging markers, indicates that the two Medicago genomes are highly similar and establishes the basis for a Medicago composite map.
    A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses
    Lévy, J. ; Bres, C. ; Geurts, R. ; Chalhoub, B. ; Kulikova, O. ; Duc, G. ; Journet, E.P. ; Ané, J.M. ; Lauber, E. ; Bisseling, T. ; Dénarié, J. ; Rosenberg, C. ; Debellé, F. - \ 2004
    Science 303 (2004)5662. - ISSN 0036-8075 - p. 1361 - 1364.
    nod factor transduction - medicago-truncatula - calcium spiking - nodulation - gene - responses - mutants - signal - recognition - perception
    Legumes can enter into symbiotic relationships with both nitrogen-fixing bacteria ( rhizobia) and mycorrhizal fungi. Nodulation by rhizobia results from a signal transduction pathway induced in legume roots by rhizobial Nod factors. DMI3, a Medicago truncatula gene that acts immediately downstream of calcium spiking in this signaling pathway and is required for both nodulation and mycorrhizal infection, has high sequence similarity to genes encoding calcium and calmodulin-dependent protein kinases (CCaMKs). This indicates that calcium spiking is likely an essential component of the signaling cascade leading to nodule development and mycorrhizal infection, and sheds light on the biological role of plant CCaMKs.
    Selection arena in Aspergillus nidulans
    Bruggeman, J. ; Debets, A.J.M. ; Hoekstra, R.F. - \ 2004
    Fungal Genetics and Biology 41 (2004)2. - ISSN 1087-1845 - p. 181 - 188.
    double-stranded-rna - sexual reproduction - podospora-anserina - neurospora-crassa - fruit abortion - seed abortion - mate choice - mutants - recombination - transmission
    The selection arena hypothesis states that overproduction of zygotes-a widespread phenomenon in animals and plants-can be explained as a mechanism of progeny choice. As a similar mechanism, the ascomycetous fungus Aspergillus nidulans may overproduce dikaryotic fruit initials, hereafter called dikaryons. Then, progeny choice might involve selection on which of these dikaryons will thrive to produce thousands of zygotes. These zygotes each produce eight sexual spores which together fill up one fruiting body. In this study, we test the selection arena hypothesis in this homothallic fungus that produces both sexual and asexual spores. We analyzed two mitochondrial and 15 auxotrophic mutations for consequences on sexual and asexual reproduction. We found that many of these mutations confer sexual self-sterility as a pleiotropic effect under conditions of normal asexual spore production. This confirms an important prediction of the selection arena, namely that dikaryons carrying a (slightly) deleterious mutation are not able to proliferate and produce sexual spores. The selection arena ensures that reproductive energy is invested mainly in dikaryons and thus sexual spores of good genetic quality. (C) 2003 Elsevier Inc. All rights reserved.
    PICKLE acts throughout the plant to repress expression of embryonic traits and may play a role in Giberellin-Dependent responses
    Henderson, J. ; Li, H.C. ; Mordhorst, A.P. ; Romero-Severson, J. ; Cheng, J.C. ; Robey, J. ; Sung, Z.R. ; Vries, S.C. de; Ogas, J. - \ 2004
    Plant Physiology 134 (2004)3. - ISSN 0032-0889 - p. 995 - 1005.
    arabidopsis seed-germination - thaliana l heynh - histone deacetylase - somatic embryogenesis - signal-transduction - leafy cotyledon1 - gene-expression - complex - mutants - locus
    A seed marks the transition between two developmental states; a plant is an embryo during seed formation, whereas it is a seedling after emergence from the seed. Two factors have been identified in Arabidopsis that play a role in establishment of repression of the embryonic state: PKL (PICKLE), which codes for a putative CHD3 chromatin remodeling factor, and gibberellin (GA), a plant growth regulator. Previous observations have also suggested that PKL mediates some aspects of GA responsiveness in the adult plant. To investigate possible mechanisms by which PKL and GA might act to repress the embryonic state, we further characterized the ability of PKL and GA to repress embryonic traits and reexamined the role of PKL in mediating GA-dependent responses. We found that PKL acts throughout the seedling to repress expression of embryonic traits. Although the ability of pkl seedlings to express embryonic traits is strongly induced by inhibiting GA biosynthesis, it is only marginally responsive to abscisic acid and SPY (SPINDLY), factors that have previously been demonstrated to inhibit GA-dependent responses during germination. We also observed that pkl plants exhibit the phenotypic hallmarks of a mutation in a positive regulator of a GA response pathway including reduced GA responsiveness and increased synthesis of bioactive GAs. These observations indicate that PKL may mediate a subset of GA-dependent responses during shoot development.
    Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation
    Palumbo, E. ; Favier, C.F. ; Deghorain, M. ; Cocconcelli, P.S. ; Grangette, C. ; Mercenier, A.M.E. ; Vaughan, E.E. ; Hols, P. - \ 2004
    FEMS Microbiology Letters 233 (2004)1. - ISSN 0378-1097 - p. 131 - 138.
    lactic-acid bacteria - lipoteichoic acid - bacillus-subtilis - escherichia-coli - teichoic-acid - lysis - methicillin - autolysis - mutants - impact
    A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation Of D-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged D-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution. (C) 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
    Analysis of natural allelic variation of Arabidopsis seed germination and seed longevity traits between the accessions Landberg erecta and Shakdara, using a new recombinant inbred line population
    Clerkx, E.J.M. ; El-Lithy, M.E.M. ; Vierling, E. ; Ruijs, G.J. ; Vries, M.H.C. de; Groot, S.P.C. ; Vreugdenhil, D. ; Koornneef, M. - \ 2004
    Plant Physiology 135 (2004)1. - ISSN 0032-0889 - p. 432 - 443.
    abscisic-acid - desiccation tolerance - molecular dissection - barley aleurone - cell-death - thaliana - loci - dormancy - mutants - quality
    Quantitative trait loci (QTL) mapping was used to identify loci controlling various aspects of seed longevity during storage and germination. Similar locations for QTLs controlling different traits might be an indication for a common genetic control of such traits. For this analysis we used a new recombinant inbred line population derived from a cross between the accessions Landsberg erecta (Ler) and Shakdara (Sha). A set of 114 F9 recombinant inbred lines was genotyped with 65 polymerase chain reaction-based markers and the phenotypic marker erecta. The traits analyzed were dormancy, speed of germination, seed sugar content, seed germination after a controlled deterioration test, hydrogen peroxide (H2O2) treatment, and on abscisic acid. Furthermore, the effects of heat stress, salt (NaCl) stress, osmotic (mannitol) stress, and natural aging were analyzed. For all traits one or more QTLs were identified, with some QTLs for different traits colocating. The relevance of colocation for mechanisms underlying the various traits is discussed.
    Modulation of the cellulose content of tuber cell walls by antisense expression of different potato (Solanum tuberosum L.) CesA clones
    Oomen, R.J.F.J. ; Tzitzikas, E. ; Bakx, E.J. ; Straatman-Engelen, I. ; Bush, M.S. ; Mccann, M.C. ; Schols, H.A. ; Visser, R.G.F. ; Vincken, J.P. - \ 2004
    Phytochemistry 65 (2004)5. - ISSN 0031-9422 - p. 535 - 546.
    synthase gene-expression - acetobacter-xylinum - catalytic subunit - infrared-spectra - arabidopsis - polysaccharides - granule - mutants - plants - resistance
    Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis. (C) 2004 Elsevier Ltd. All rights reserved.
    The role of cryptochrome 2 in flowering in Arabidopsis
    El-Assal, S.E.D. ; Alonso-Blanco, C. ; Peeters, A.J.M. ; Wagemaker, C. ; Weller, J.L. ; Koornneef, M. - \ 2003
    Plant Physiology 133 (2003)4. - ISSN 0032-0889 - p. 1504 - 1516.
    landsberg erecta - phytochrome-a - circadian clock - locus-c - genetic-control - time - thaliana - mutants - encodes - phenotype
    We have investigated the genetic interactions between cry2 and the various flowering pathways in relation to the regulation of flowering by photoperiod and vernalization. For this, we combined three alleles of CRY2, the wild-type CRY2-Landsberg erecta (Ler), a cry2 loss-of-function null allele, and the gain-of-function CRY2-Cape Verde Islands (Cvi), with mutants representing the various photoreceptors and flowering pathways. The analysis of CRY2 alleles combined with photoreceptor mutants showed that CRY2-Cvi could compensate the loss of phyA and cry1, also indicating that cry2 does not require functional phyA or cry1. The analysis of mutants of the photoperiod pathway showed epistasis of co and gi to the CRY2 alleles, indicating that cry2 needs the product of CO and GI genes to promote flowering. All double mutants of this pathway showed a photoperiod response very much reduced compared with Ler. In contrast, mutations in the autonomous pathway genes were additive to the CRY2 alleles, partially overcoming the effects of CRY2-Cvi and restoring day length responsiveness. The three CRY2 alleles were day length sensitive when combined with FRI-Sf2 and/or FLC-Sf2 genes, which could be reverted when the delay of flowering caused by FRI-Sf2 and FLC-Sf2 alleles was removed by vernalization. In addition, we looked at the expression of FLC and CRY2 genes and showed that CRY2 is negatively regulated by FLC. These results indicate an interaction between the photoperiod and the FLC-dependent pathways upstream to the common downstream targets of both pathways, SOC1 and FT.
    Vegetative compatibility groups in Verticillium dahliae isolates from the Netherlands as compared to VCG diversity in Europe and in the USA
    Hiemstra, J.A. ; Rataj-Guranowska, M. - \ 2003
    European Journal of Plant Pathology 109 (2003)8. - ISSN 0929-1873 - p. 827 - 839.
    genetic-relationships - fusarium-oxysporum - tester strains - pathogenicity - virulence - mutants - cotton - soil - differentiation - populations
    In a study of vegetative compatibility in Verticillium dahliae in the Netherlands, a collection of 45 isolates including representatives from woody hosts, several horticultural crops and from the soil of potato fields was examined. In addition an effort was made to compare vegetative compatibility groups (VCGs) from different countries. The results of this study indicate that VCG diversity in V. dahliae in the Netherlands is limited. Only two VCGs were detected: VCG NL-I and VCG NL-II. The former is the predominant VCG for isolates from tree hosts. However, Verticillium wilt in trees can be caused by isolates from both VCGs. It is suggested that the predominance of VCG NL-I in tree hosts is the result of the origin of the tree and the cropping history of its growing site, rather than trees being preferential hosts for isolates from this VCG. Comparison of VCG testers from the Netherlands, from several other European countries and from the USA show that in Europe two major VCGs are present. The first one, including NL-I, is compatible with USA VCG 3 and VCG 4, whereas the second one, including NL-II, is compatible with USA VCG 1 and VCG 2. These groups are not completely separated; in some cases, testers formed heterokaryons with VCG testers from both main groups. Because of the presence of these bridge isolates and because mutants from the same isolate differ in ability to form heterokaryons, it is emphasised that careful selection of isolate testers is an essential step to get a clear picture of VCG diversity.
    An integrated physical, genetic and cytogenetic map around the sunn locus of Medicago truncatula
    Schnabel, E. ; Kulikova, O. ; Penmetsa, R.V. ; Bisseling, T. ; Cook, D.R. ; Frugoli, J. - \ 2003
    Genome 46 (2003)4. - ISSN 0831-2796 - p. 665 - 672.
    expressed sequence tags - nod factor transduction - legume lotus-japonicus - receptor-like kinase - root-hair - model - nodulation - mutants - fish - pachytene
    The sunn mutation of Medicago truncatula is a single-gene mutation that confers a novel supernodulation phenotype in response to inoculation with Sinorhizobium meliloti. We took advantage of the publicly available codominant PCR markers, the high-density genetic map, and a linked cytogenetic map to define the physical and genetic region containing sunn. We determined that sunn is located at the bottom of linkage group 4, where a fine-structure genetic map was used to place the locus within a similar to400-kb contig of bacterial artificial chromosome (BAC) clones. Genetic analyses of the sunn contig, as well as of a second, closely linked BAC contig designated NUM1, indicate that the physical to genetic distance within this chromosome region is in the range of 1000 -1100 kb.cM(-1). The ratio of genetic to cytogenetic distance determined across the entire region is 0.3 cM-mum(-1). These estimates are in good agreement with the empirically determined value of similar to300 kb-mum(-1) measured for the NUM1 contig. The assignment of sunn to a defined physical interval should provide a basis for sequencing and ultimately cloning the responsible gene.
    Metabolic engineering of the carotenoid biosynthetic pathway in the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma)
    Verdoes, J.C. ; Sandmann, G. ; Visser, H. ; Diaz, M. ; Mossel, M. van; Ooyen, A.J.J. van - \ 2003
    Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 3728 - 3738.
    escherichia-coli - astaxanthin production - beta-carotene - gene - mutants - microorganisms - optimization - expression - mice
    The crtYB locus was used as an integrative platform for the construction of specific carotenoid biosynthetic mutants in the astaxanthin-producing yeast Xanthophyllomyces dendrorhous. The crtYB gene of X. dendrorhous, encoding a chimeric carotenoid biosynthetic enzyme, could be inactivated by both single and double crossover events, resulting in non-carotenoid-producing transformants. In addition, the crtYB gene, linked to either its homologous or a glyceraldehyde-3-phosphate dehydrogenase promoter, was overexpressed in the wild type and a beta-carotene-accumulating mutant of X. dendrorhous. In several transformants containing multiple copies of the crtYB gene, the total carotenoid content was higher than in the control strain. This increase was mainly due to an increase of the beta-carotene and echinone content, whereas the total content of astaxanthin was unaffected or even lower. Overexpression of the phytoene synthase-encoding gene (crtI) had a large impact on the ratio between mono- and bicyclic carotenoids. Furthermore, we showed that in metabolic engineered X. dendrorhous strains, the competition between the enzymes phytoene desaturase and lycopene cyclase for lycopene governs the metabolic flux either via beta-carotene to astaxanthin or via 3,4-didehydrolycopene to 3-hydroxy-3'-4'-didehydro-beta-psi-caroten-4-one (HDCO). The monocylic carotenoid torulene and HDCO, normally produced as minority carotenoids, were the main carotenoids produced in these strains.
    Analysis of natural allelic variation at seed dormancy loci of Arabidopsis thaliana
    Alonso-Blanco, C. ; Bentsink, L. ; Hanhart, C.J. ; Vries, M.H.C. de; Koornneef, M. - \ 2003
    Genetics 164 (2003)2. - ISSN 0016-6731 - p. 711 - 729.
    quantitative trait loci - flowering time - abscisic-acid - l heynh - linkage map - germination - mutants - gibberellin - gene - light
    Arabidopsis accessions differ largely in their seed dormancy behavior. To understand the genetic basis of this intraspecific variation we analyzed two accessions: the laboratory strain Landsberg erecta (Ler) with low dormancy and the strong-dormancy accession Cape Verde Islands (Cvi). We used a quantitative trait loci (QTL) mapping approach to identify loci affecting the after-ripening requirement measured as the number of days of seed dry storage required to reach 50% germination. Thus, seven QTL were identified and named delay of germination (DOG) 1-7. To confirm and characterize these loci, we developed 12 near-isogenic lines carrying single and double Cvi introgression fragments in a Ler genetic background. The analysis of these lines for germination in water confirmed four QTL (DOG1, DOG2, DOG3, and DOG6) as showing large additive effects in Ler background. In addition, it was found that DOG1 and DOG3 genetically interact, the strong dormancy determined by DOG1-Cvi alleles depending on DOG-3-Ler alleles. These genotypes were further characterized for seed dormancy/germination behavior in five other test conditions, including seed coat removal, gibberellins, and an abscisic acid biosynthesis inhibitor. The role of the Ler/Cvi allelic variation in affecting dormancy is discussed in the context of current knowledge of Arabidopsis germination.
    Characterization of green seed, an enchancer of abi3-1 in Arabidopsis that affects seed longevity
    Clerkx, E.J.M. ; Vries, M.H.C. de; Ruijs, G.J. ; Groot, S.P.C. ; Koornneef, M. - \ 2003
    Plant Physiology 132 (2003)2. - ISSN 0032-0889 - p. 1077 - 1084.
    biologische landbouw - zaadbehandeling - zaadlevensduur - zaadfysiologie - opslag van zaden - organic farming - seed treatment - seed longevity - seed physiology - seed storage - abscisic-acid biosynthesis - chlorophyll degradation - protein accumulation - signal-transduction - stay-green - mutants - thaliana - quality - gene - deterioration
    Seeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germination
    Seeds are usually stored in physiological conditions in which they gradually lose their viability and vigor depending on storage conditions, storage time, and genotype. Very little is known about the underlying genetics of seed storability and seed deterioration. We analyzed a mutant in Arabidopsis disturbed in seed storability. This mutant was isolated as a grs (green-seeded) mutant in an abi3-1 (abscisic acid 3) mutant background. Genetic and physiological characterization showed that the monogenic grs mutant was not visibly green seeded and mapped on chromosome 4. This enhancer mutation did not affect the ABA sensitivity of seed germination or seed dormancy but was found to affect seed storability and seedling vigor. Seed storability was assessed in a controlled deterioration test, in which the germination capacity of the mutant decreased with the duration of the treatment. The decrease in viability and vigor was confirmed by storing the seeds in two relative humidities (RHs) for a prolonged period. At 60% RH, the mutant lost germinability, but storage at 32% RH showed no decrease of germination although seed vigor decreased. The decrease in viability and vigor could be related to an increase in conductivity, suggesting membrane deterioration. This was not affected by light conditions during imbibition, expected to influence the generation of active oxygen species. During seed maturation, ABI3 regulates several processes: acquiring dormancy and long-term storability and loss of chlorophyll. Our results indicate that GRS is a common regulator in the latter two but not of dormancy/germination.
    Quantification of disease progression of several microbial pathogens on Arabidopsis thaliana using real-time fluorescence PCR
    Brouwer, M. ; Lievens, B. ; Hemelrijck, W. van; Ackerveken, G. van den; Cammue, B.P.A. ; Thomma, B.P.H.J. - \ 2003
    FEMS Microbiology Letters 228 (2003)2. - ISSN 0378-1097 - p. 241 - 248.
    expressing beta-glucuronidase - polymerase-chain-reaction - alternaria-brassicicola - fungal biomass - resistance - infection - assay - gene - susceptibility - mutants
    An accurate monitoring of disease progression is important to evaluate disease susceptibility phenotypes. Over the years, Arabidopsis thaliana has become the model species to serve as a host in plant-pathogen interactions. Despite the efforts to study genetic mechanisms of host defense, little efforts are made for a thorough pathogen assessment, often still depending on symptomology. This manuscript describes the use of real-time polymerase chain reaction (PCR) to assess pathogen growth in the host Arabidopsis for a number of frequently studied pathogens. A wide range of correlations between pathogen biomass and fluorescence is demonstrated, demonstrating the theoretical sensitivity of the technique. It is also demonstrated that host DNA does not interfere with the quantification of pathogen DNA over a wide range. Finally, quantification of pathogen biomass in different plant genotypes with a varying degree of resistance shows the capability of this technique to be used for assessment of pathogen development in disease progression. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
    Male and female roles in crosses of Aspergillus nidulans as revealed by vegetatively incompatible parents
    Bruggeman, I.M. ; Debets, A.J.M. ; Swart, K. ; Hoekstra, R.F. - \ 2003
    Fungal Genetics and Biology 39 (2003). - ISSN 1087-1845 - p. 136 - 141.
    heterokaryon incompatibility - neurospora-crassa - inheritance - evolution - mutants - meiosis - genome - nuclei
    To resolve the role of male and female nuclei and mitochondria in cleistothecium formation in the model organism Aspergillus nidulans, we analysed the genetic constituents of cleistothecia, from crosses between vegetatively compatible and incompatible parents. We used markers that enabled us to determine the nuclear genotype of the cleistothecial wall and the nuclear and mitochondrial genotype of the ascospores. In compatible parents, nuclear genomes and cytoplasm usually mix in the vegetative hyphae prior to the formation of the sexual stage after which any cleistothecial composition is possible. In incompatible parents, the maternal strain contributes the nuclei for the cleistothecial wall and one nucleus as well as mitochondria for the ascospore origin. The paternal strain donates one nucleus for the ascospore origin. Only in crosses between vegetatively incompatible partners, it is possible to assign a female and male role to the parental strains. Our results confirm that the vegetative heterokaryotic stage is not a prerequisite for cleistothecium formation. Using this tool, we analysed sexual sporulation mutants for male or female sterility. (C) 2003 Elsevier Science (USA). All rights reserved.
    Verslag Boskoopmutantenproef Horst en Wilhelminadorp : 069-9502
    Steeg, P.A.H. van der; Kemp, H. - \ 2002
    Randwijk : Praktijkonderzoek Plant & Omgeving, Sector Fruit (Rapport / Praktijkonderzoek Plant & Omgeving, Sector Fruit nr. 2002-1) - 43
    appels - mutanten - gebruikswaarde - kwaliteit - plantenveredeling - apples - mutants - use value - quality - plant breeding
    Dit onderzoek is gefinacierd door het productschap Tuinbouw
    Verslag mutantenproeven Cox's Orange Pepin en Queen Cox in Wilhelminadorp : 069 Wi 9102, -9205, -9206, -9207
    Kemp, H. ; Steeg, P.A.H. van der - \ 2002
    Randwijk : Praktijkonderzoek Plant & Omgeving, Sector Fruit (Rapport / Praktijkonderzoek Plant & Omgeving, Sector Fruit nr. 2002-8) - 69
    appels - mutanten - plantenveredeling - experimenten - apples - mutants - plant breeding - experiments
    Onderzoek door PT, Produktschap Tuinbouw gefinancierd
    Verslag Elstarmutantenproef 069 Ze 9506
    Kemp, H. ; Steeg, P.A.H. van der - \ 2002
    Randwijk : Praktijkonderzoek Plant & Omgeving, Sector Fruit (Rapport / Praktijkonderzoek Plant & Omgeving, Sector Fruit nr. 2002-16) - 24
    malus pumila - appels - cultivars - rassen (planten) - mutanten - rassenproeven - nederland - malus pumila - apples - cultivars - varieties - mutants - variety trials - netherlands
    Dit onderzoek is gefinancierd door het Productschap Tuinbouw
    Timeline - A fortunate choice: the history of Arabidopsis as a model plant
    Somerville, C. ; Koornneef, M. - \ 2002
    Nature Reviews Genetics 3 (2002)11. - ISSN 1471-0056 - p. 883 - 889.
    thaliana l heynh - artificial chromosome library - encoding nitrate reductase - polymorphism linkage map - floral homeotic genes - agrobacterium-tumefaciens - mediated transformation - dna - mutants - sequence
    During the past 20 years, the flowering plant Arabidopsis thaliana has been adopted as a model organism by thousands of biologists. This community has developed important tools, resources and experimental approaches that have greatly stimulated plant biological research. Here, we review some of the key events that led to the uptake of Arabidopsis as a model plant and to the growth of the Arabidopsis community.
    Classical mutagenesis in higher plants
    Koornneef, M. - \ 2002
    In: Molecular Plant Biology / Gilmartin, P.M., Bowler, C., Oxford, GB : Oxford University Press - p. 1 - 10.
    genetica - mutagenese - mutanten - plantenveredeling - mutagenen - genetics - mutagenesis - mutants - plant breeding - mutagens
    For a long time, mutagenesis research in plants focused on crop improvement and, especially for crop plants, opimised protocols were developed with barley being one of the favourite species. However, the interest in mutagenesis has shifted to basic plant research in the last 20 years, when the power of mutant approaches in combination with molecular techniques to investigate the molecular nature of the genes became fully appreciated
    Two approaches for induction and isolation of starch mutants in potato (Solanum tuberosum L.): random versus gene targeted mutagenesis = Twee benaderingen voor de inductie en isolatie van zetmeelmutanten in aardappel (Solanum tuberosum L.): ongerichte versus gen gerichte mutagenese
    Hoogkamp, T.J.H. - \ 2001
    Wageningen University. Promotor(en): E. Jacobsen; R.G.F. Visser. - S.l. : S.n. - ISBN 9789058084538 - 92
    aardappelen - solanum tuberosum - plantenveredeling - geïnduceerde mutaties - zetmeel - transposons - haploïdie - mutanten - transpositie - potatoes - solanum tuberosum - plant breeding - induced mutations - starch - transposable elements - haploidy - mutants - transposition

    In this thesis two approaches were used to induce structural mutations in potato starch biosynthesis genes in potato. First production of new monoploid amf genotypes through parthenogenesis made it possible to initiate mutation breeding for amfae double mutants. Two amf monoploids were selected which fulfilled most of the prerequisites. By inducing a mutation in one of the branching enzymes in an amf -mutant it is possible to select a double mutant having less branched amylopectin. This mutation can easily be identified by iodine staining. Amylose-free starch will stain red and less branched amylopectin will stain blue, like amylose containing starch. Mutations were induced by X-ray irradiation of leaf explants followed by adventitious shoot regeneration and microtuber induction or followed by several rounds of multiplication of axillary buds and microtuber induction. In both cases the starch of microtubers was stained with iodine to screen for aberrant types. In 56 tuber samples blue or otherwise aberrant starch granules were found. With this kind of observations, the concept of mutation breeding for starch variants in monoploid potatoes is proven. A second way to induce structural mutations was the use of the Ac ( Activator )/ Ds ( Dissociation ) transposase system of maize in potato where the Ds transposon is activated by a transposase source. In this study the Ds element was linked to the GBSS gene, of which the phenotypic effect of deactivation is known i.e. red staining starch after iodine staining. The known amf mutation was used as a model system to gather more information about the transposition frequency of the Ds transposable elements in potato and to test the tagging of the wildtype GBSS gene. To activate the Ds element four Ds transposon containing plants were combined with the Ac transposase via cross combination or double transformation. Excision rates ranged from 14.8-48.4 %. Three phenotypic starch mutants were found after screening by iodine staining of tuber cut surfaces. These amylose-free mutants were analyzed by in vitro tests, Southern blot hybridization and sequencing. Strong indications were found that inactivation of the GBSS gene was caused by a transposable element.

    Meiotic sister chromatid cohesion and recombination in two filamentous fungi
    Heemst, D. van - \ 2000
    Agricultural University. Promotor(en): C. Heyting; H.W.J. van den Broek. - S.l. : S.n. - ISBN 9789058083180 - 127
    moleculaire genetica - meiose - chromatiden - zusterchromatidenuitwisseling - recombinatie - schimmels - emericella nidulans - pezizomycotina - mutanten - dna-sequencing - molecular genetics - meiosis - chromatids - sister chromatid exchange - recombination - fungi - emericella nidulans - pezizomycotina - mutants - dna sequencing

    Homologous recombination and sister chromatid cohesion play important roles in the maintenance of genome integrity and the fidelity of chromosome segregation in mitosis and meiosis. Within the living cell, the integrity of the DNA is threatened by various factors that cause DNA-lesions, of which DNA double-strand breaks (DSBs) are considered particularly deleterious. The causative agents can be of endogenous origin, such as metabolically produced free radicals, and of exogenous origin, such as ultraviolet light and ionizing radiation. The accurate repair of DSBs is important to prevent chromosomal fragmentation, translocations and deletions. Of the sophisticated (networks of) DNA repair pathways that have evolved, homologous recombination, which repairs the DSB by copying information from an intact homologous DNA-template, is considered one of the most accurate. In mitotic G2, the sister chromatid is preferentially used as a template for recombinational repair of DSBs.

    DSBs can also arise as normal intermediates in several DNA repair and recombination pathways, including meiotic recombination. In meiotic recombination (in yeast), the cells actively induce large numbers of DSBs, and channel their search for a homologous template towards a non-sister chromatid of the homologous chromosome. In each pair of homologous chromosomes, at least one DSB is repaired by reciprocal exchange of precisely corresponding segments of non-sister chromatids (crossing over), whereas additional DSBs in the same chromosome pair are repaired by either reciprocal or non-reciprocal exchange. The reciprocal exchanges between non-sister chromatids (visible as chiasmata) are essential for the proper disjunction of homologous chromosomes during the first meiotic division (meiosis I).

    When I started my investigations for this thesis, it was recognized that reciprocal exchanges as such could not direct the proper segregation of homologous chromosomes at meiosis I; some "glue" should keep the chiasmata in place, either by binding to the chiasmata, or by maintaining cohesion between the sister chromatids distal to the chiasmata. Although mutants existed that appeared to be defective in the production of this glue, its nature remained unknown. In this thesis, I have tried to identify components involved in meiotic sister chromatid cohesion and recombination, to analyze the interplay between these two processes in meiosis and to gain insight into their relationship with mitotic DNA repair and recombination.

    In chapter 1 , I explain the choice of the experimental model systems that I used for the research described in this thesis. All investigations were performed in two filamentous fungi, namely Sordaria macrospora and Aspergillus nidulans . The most important reason for the choice of these two fungi was that mutants were available (or easily obtainable) that were defective in meiotic sister chromatid cohesion and/or recombination, and that it should be feasible to clone the corresponding wild-type genes by means of transformation complementation of the mutant defects. S. macrospora had the additional advantage of a well-developed cytology and A. nidulans had the additional advantages of well-developed molecular genetic tools and the presence of a parasexual cycle in addition to the sexual cycle. This latter feature offers the possibility of analyzing mitotic allelic recombination. Moreover, A. nidulans is one of the two known organism that do not assemble synaptonemal complexes (SCs) during meiotic prophase and that do not display positive crossover interference. The choice for both S. macrospora and A. nidulans would thus make it possible to compare the role(s) of genes involved in meiotic sister chromatid cohesion and/or recombination in a organism with and one without SCs.

    In chapter 2 , we describe the cloning of the SPO76 gene of S. macrospora by transformation complementation of the meiotic defects of the spo76-1 (non-null) mutant. It was known that this mutant displayed defects in meiotic sister chromatid cohesion, meiotic recombination and mitotic DNA-repair. Furthermore, we analyzed the localization of the Spo76 protein throughout wild-type mitosis and meiosis and performed a detailed analysis of the spo76-1 mutant phenotype. We show that Spo76p is chromosome-associated during all stages of mitosis and meiosis, except at metaphase(s) and anaphase(s). During mitosis, Spo76p disappears from the chromosomes at prometaphase. During meiotic prophase I, Spo76p is more abundant than during any other cell cycle stage, and localizes preferentially close to the chromosome axes. Spo76p disappears from the chromosomes at diplotene. In the spo76-1 mutant, we observed a transient defect in chromosome organization at (mitotic) prometaphase: the duration of this stage was prolonged and chromosome morphology was abnormal. Strikingly, chromosomal regions with defects in both sister chromatid cohesion and chromosome compaction alternated with regions with apparently normal cohesion and compaction. We speculate that the mitotic prophase to metaphase transition involves forces that tend to disrupt cohesion and that Spo76p promotes the maintenance of a minimum of cohesion in combination with chromosome compaction. Likewise, we observed in meiotic prophase of spo76-1 , from late leptotene on, that sister chromatid cohesion and chromosome compaction were coordinately affected on a regional basis. Regions with widely split axial elements alternated with regions with unsplit segments of axial elements. These unsplit segments could form stretches of SC, which contained rare late recombination nodules (late RNs: ultrastructurally recognizable enzyme-complexes involved in the later steps of meiotic recombination). Whereas the number of late RNs was strongly reduced in spo76-1 , early RNs (as recognized by immunocytochemical labelling of Rad51 and Dmc1) occurred at only slightly reduced levels in the mutant and persisted longer. This suggest that spo76-1 is deficient in some intermediate step of meiotic recombination. The role of Spo76p in the meiotic leptotene/zygotene transition may be related to its role during the mitotic prophase/metaphase transition, in that the leptotene/zygotene transition might also bring along forces that tend to disrupt cohesion, and that Spo76p is also needed for maintenance of cohesion during this meiotic transition. Spo76p may have an additional role in late meiotic prophase because in spo76-1 , meiotic sister chromatids separated completely from diplotene on, whereas in wild type this does not occur before anaphase II.

    The predicted protein encoded by the SPO76 gene is evolutionary conserved from fungi to man; the highest percentage of amino acid identity (44%) was found with the BIMD protein of A. nidulans. The A. nidulansbimD6 mutant was previously identified as a conditional lethal mutant with a (lethal) mitotic chromosome segregation defect at high temperature and a (non-lethal) DNA repair deficiency phenotype at low temperature.

    In chapter 3 , we demonstrate by heterologous complementation that the SPO76 gene of S. macrospora can complement both the temperature and the MMS (methyl methane sulphonate) sensitivities of bimD6 in A. nidulans , implying direct functional homology between the mitotic roles of two proteins. We also show that, like spo76-1 , bimD6 mutants do not form sexual spores (ascospores). However, bimD6 mutants display, unlike spo76-1 , disturbances in premeiotic development and form only few asci. In the few asci that entered meiosis, sister chromatids separate prematurely, but the extent of meiotic sister chromatid separation is less severe in bimD6 than in spo76-1 . In addition, whereas the mitotic localization of the two proteins was roughly similar, the meiotic localization of the two proteins showed some important differences. Unlike Spo76p in S. macrospora , BIMD in A. nidulans was not more abundant during meiotic prophase than in the mitotic cycle, and did not localize preferentially close to the chromosome axes during the pairing of homologous chromosomes. Moreover, SPO76 could not complement the sexual sporulation defects of bimD6 in A. nidulans, and vice versa , BIMD could not restore the sexual sporulation defects of the S. macrospora spo76-1 mutant. These results indicate that Spo76p and BIMD may differ in a species-specific manner with respect to their meiotic roles. The species-specific aspects of the roles of Spo76p and BIMD6 in meiosis are possibly related to the differences in meiotic chromosome organization between the two fungi (see above; chapter 1 ): in contrast to S. macrospora , A. nidulans does not form SCs and does not display positive interference of meiotic crossovers.

    We also show that bimD6 mutants are hypersensitive to X-rays in addition to their elevated sensitivities to UV and MMS, but only when dividing cells are exposed to these agents. The bimD6 mutant thus closely resembles recombination-deficient mutants of A. nidulans , such as uvsC114 (the uvsC gene of A. nidulans is homologous to RAD51 of Saccharomyces cerevisiae ; chapter 4 ). We have therefore compared bimD6 with uvsC114 regarding defects in mitotic recombination. When assayed for allelic recombination, bimD6 and uvsC114 yielded similar results. In both mutants, the absolute frequencies of allelic recombination were strongly reduced, although the distribution of recombinants among the various classes was comparable to wild type. However, when assayed for intrachromosomal conversions, bimD6 and uvsC114 produced different results. Intrachromosomal conversions between interrupted duplications were significantly reduced in uvsC114 , but occurred at wild-type frequencies in bimD6 . We propose that BIMD is required for homologous recombination when the template is located on another (sister or non-sister) chromatid, but not when the template is available in close proximity on the same sister chromatid or the same chromatin domain/loop. The repair machinery may thus be obliged to cooperate with cohesion complexes (which are probably located at the borders between chromatin domains/loops) if the homologous template lies outside an intrachromatid loop domain. In contrast, uvsC was required for both types of repair.

    In chapter 4 , we describe the cloning of the uvsC gene of A. nidulans by transformation complementation of the mitotic repair defects of a uvsC114 mutant. Furthermore, we disrupted the entire uvsC gene, and we compared the phenotypic effects of the resulting null mutation with those of uvsC114 . The predicted UVSC protein shows 67% amino acid identity with Rad51p of S. cerevisiae and 27% amino acid identity with the RecA protein of Escherichia coli. These proteins are involved in strand invasion and exchange during homologous recombination. We found that in the absence of DNA-damaging agents, the uvsC gene was transcribed at a higher level in the uvsC114 mutant than in wild-type. Transcription of uvsC was inducible by MMS in wild-type and uvsC114 mutant strains. We compared the mitotic and meiotic phenotypes of mutants carrying the uvsC114 point mutation (a deletion of 6 bp in core domain I) with those of the uvsC null mutant. The uvsC null mutant was more sensitive to UV and MMS than uvsC114 , indicating that uvsC114 is not a null mutation. The sexual developmental phenotypes of the two mutants also differed. In the uvsC null mutant, sexual development was disturbed before the onset of meiosis, whereas in uvsC114 it was blocked in meiotic prophase. We observed large, multi-nucleated cells in older cleistothecia of the uvsC null mutant These cells possibly represent degenerated croziers, which might have been blocked at premeiotic S-phase. In S. cerevisiae , disruption of RAD51 has no effect on mitotic growth and causes arrest at meiotic prophase I, whereas in mouse, disruption of Rad51 results in embryonic lethality. In A. nidulans , disruption of uvsC had no effect on mitotic growth, and caused an arrest at a premeiotic stage of sexual development. Disruption of RAD51 -homologous genes thus has different effects in different organisms.

    In chapter 5 , we describe the isolation and characterization of sexual sporulation mutants of A. nidulans . Vegetative spores were treated with a high dose of UV (1.5 % survival) and surviving colonies were plated on supplemented minimal medium. Colonies that did not display aberrant vegetative growth were visually screened for the appearance of "barren" fruiting bodies (=fruiting bodies without or with only few ascosores). This screen (1250 colonies analyzed) yielded 20 mutants with the desired phenotype. After two successive rounds of backcrosses with wildtype, two mutants yielded no longer progeny with the original mutant phenotype; these mutants were not analyzed further. The remaining 18 mutants were all recessive and were assigned to 15 complementation groups. Based on these numbers, we estimate that, under the growth conditions tested, about 50-100 genes are specifically involved in ascospore formation in A. nidulans . Three mutations were mapped by parasexual analysis: two mutations could be assigned to a specific chromosome, and one was associated with a translocation breakpoint. For all 18 mutants, the contents of the "barren" fruiting bodies were cytologically analyzed. A large proportion of the mutants, namely 11 out of 18, arrested in meiotic prophase I (like uvsC114 ; chapter 4 ) or metaphase I (like bimD6 ; chapter 3 ). It is thus possible that this new collection contains mutants that are specifically affected in meiotic recombination and/or sister chromatid cohesion. We suggest a strategy to clone the corresponding wild-type genes, by selecting for the appearance of prototrophic progeny clones that result from meiotic reassortment of auxotrophic markers.

    In the General Discussion ( chapter 6) , we speculate upon the possible links between sister chromatid cohesion and recombination in mitosis and meiosis. Whereas in mitosis centromeric cohesion primarily serves chromosome disjunction, arm cohesion may play additional roles in repair by recombination. Cohesion complexes at the basis of the chromatin domains/loops may function as nucleation sites for the assembly of recombinational repair complexes and assist these complexes in finding a homologous template in a precisely corresponding segment of the undamaged sister chromatid. They may thus be responsible for the observed bias for the sister chromatid as a template for recombinational repair during mitotic G2. In meiosis I, recombination has to be directed towards a non-sister chromatid of the homologous chromosome and, at the same time, arm cohesion has to be maintained and possibly even reinforced to ensure correct reductional chromosome segregation. Consequently, both the recombinational repair machinery and the cohesion complexes function in meiosis in a modified form. In this modified form, cohesion complexes may serve as a basis for axial element formation. DSBs are probably produced concomitantly with axial element assembly. As we proposed for mitotic recombinational repair, we hypothesize that meiotic DSBs are transferred to the basis of chromatin loops/domains, where they are brought into contact with the meiotic cohesion complexes and additional proteins required for recombinational repair. However, we speculate that homology search on the sister chromatid will now be blocked by linear element components so that another template for homologous recombination has to be found. Furthermore, axial element components, in concert with the modified recombination complex, will possibly establish DNA-DNA contacts with a non-sister chromatid of the homologous chromosome. Thus, by providing the basis for linear element formation and assembly of a meiosis-specific recombination complex, cohesion complexes may contribute to the preference for a non-sister chromatid of the homologous chromosome as a template for homologous recombination in meiosis.

    A genetic and molecular analysis of two genes involved in flowering initiation of Arabidopsis = [Een genetische en moleculaire analyse van twee genen die betrokken zijn bij de bloei initiatie van Arabidopsis]
    Soppe, W.J.J. - \ 2000
    Agricultural University. Promotor(en): M. Koornneef. - S.l. : S.n. - ISBN 9789058082893 - 120
    arabidopsis - arabidopsis thaliana - genetische analyse - genen - genexpressie - biotechnologie - bloei - fotoperiodiciteit - circadiaan ritme - moleculaire genetica - mutanten - genomen - arabidopsis - arabidopsis thaliana - genetic analysis - genes - gene expression - biotechnology - flowering - photoperiodism - circadian rhythm - molecular genetics - mutants - genomes

    The transition from the vegetative to the reproductive phase (flowering initiation) in plants has a complex regulation which is affected by environmental and internal plant factors. The understanding of this process is not only of fundamental interest but could also lead to practical applications. The research into flowering initiation has a long history. The initial emphasis on physiological and biochemical studies led to the identification of different factors that influence flowering time. During the sixties, a genetic approach was initiated in different plant species. In Arabidopsis several late flowering mutants were isolated and genetically and physiologically characterised which revealed a complex regulation of flowering time by different pathways. These are the photoperiodic promotion pathway which promotes flowering under long day light conditions, the vernalisation promotion pathway which promotes flowering by low temperatures and the autonomous promotion pathway which promotes flowering independent of the environment. Due to its favourable genetic and molecular features, research on flowering initiation became focussed on Arabidopsis. Since the beginning of the nineties, several of the genes involved in the different pathways have been cloned, providing more information about the function of these genes in the cell and their relations with each other. Despite this increasing amount of information, the picture is still far from complete.

    The aim of the work presented in this thesis is to increase our knowledge of flowering time regulation. It focussed on the genetic and molecular characterisation of the semi-dominant mutant fwa , which flowers late under long day light conditions and has been proposed to be part of the photoperiodic promotion pathway. One approach sought to identify additional genes that affect flowering by mutagenesis of the fwa mutant. In addition to three different intragenic revertants of fwa , this screen yielded a novel early flowering mutant.

    This mutant was named early flowering in short days ( efs ). Its phenotypic characterisation has shown that the main role of the wild-type EFS gene is to delay flowering in plants that have entered the adult vegetative phase, which is considered to be the phase where plants are able to respond to environmental signals in order to flower. Consistent with this, efs mutant plants do not show an early flowering phenotype when grown under environmental conditions that lead to a shortened adult vegetative phase such as long days and vernalisation. To learn more about the role of EFS in relation to other genes involved in flowering initiation, double mutants were isolated. Their characterisation showed that efs is involved in the autonomous promotion pathway. This result, together with the lack of a vernalisation response, suggests that EFS is likely to represent a new element acting at a point close to the convergence of signals from the autonomous promotion pathway and the vernalisation promotion pathway.

    The main topic of this thesis concerns the map based cloning of the FWA gene. By using plants which have a cross-over between FWA and surrounding markers, the FWA locus could be located in a region of about 60 Kb. Plant transformation experiments with cosmids spanning this region showed that the gene is located in the overlap of two cosmids. This overlap contained only one complete gene that encodes a homeodomain containing transcription factor. The altered expression of this gene in fwa mutants together with DNA mutations in the intragenic revertants of fwa-1 further proved that this gene is FWA .

    Analysis of FWA revealed several interesting characteristics. Surprisingly, the mutant and wild-type alleles had an identical DNA sequence in the FWA region, excluding DNA mutations in the gene as a cause for the mutant phenotype. Furthermore, two direct repeated sequences were found in the 5' genomic region of FWA . In wild-type plants these repeats were heavily methylated, whereas they were completely un-methylated in the mutant alleles. In contrast to fwa mutant plants, which showed a high expression of FWA at all developmental stages, wild-type plants showed only a low expression of FWA in siliques and germinating seeds. Taken together, these findings suggest that loss of methylation of the FWA repeats in the fwa mutant causes a high level of expression of the gene, leading to a late flowering phenotype. A similar correlation of late flowering, FWA overexpression and hypomethylation of FWA repeats was found in late flowering plants which were derived from the ddm1 hypomethylation mutant. The late flowering phenotype of these plants had previously been mapped to the FWA region. Nevertheless, the correlation between hypomethylation of the FWA repeats and FWA expression was not found in germinating seeds of wild-type plants which showed expression of FWA but methylation of the repeats. Although this expression might come from residual mRNA produced earlier in developing seeds, it is possible that methylation of the repeats does not always prevent expression of FWA . Perhaps a different epigenetic mechanism early in development can induce expression of methylated genes.

    The correlation of FWA expression with late flowering indicates that FWA is a repressor of flowering. Earlier studies had already shown that FWA does not only play a role in the initiation of flowering but also in flower meristems. However, the FWA transcript was not detected in flower buds or flowers and therefore, FWA might only affect this process when highly expressed in the fwa mutant. Possibly, FWA has no function in flowering initiation of wild-type. It might participate in a seed-specific process, as suggested by its expression in seeds. However, the lack of an obvious phenotype suggests that this role is minor or redundant with other genes.

    The cloning of FWA revealed that the absence of methylation in the repeating sequences in the 5' region of the FWA gene leads to an enhanced expression in the fwa mutant. However, it did not become clear whether this correlation is direct or indirect. Also the importance of the methylation in wild-type plants is still unclear. It is possible that it has a role in the expression of the gene under specific environmental conditions.

    The results discussed in this thesis have contributed to the existing knowledge of flowering initiation by the isolation of a mutant at a novel locus and the cloning of a previously known gene which are both involved in this process. In addition, the results suggest a possible role for DNA methylation in gene regulation of Arabidopsis.

    Outer membrane protein changes during bacteroid development are independent of nitrogen fixation and differ between indeterminate and determinate nodulating host plants of Rhizobium leguminosarum
    Roest, H.P. ; Goosen-de Roo, L. ; Wijffelman, C.A. ; Maagd, R.A. de; Lugtenberg, B.J.J. - \ 1995
    Molecular Plant-Microbe Interactions 8 (1995)1. - ISSN 0894-0282 - p. 14 - 22.
    monoclonal-antibodies - root-nodules - golgi bodies - cell-surface - sym-plasmid - lipopolysaccharide - mutants - forms - identification - expression
    The outer membrane of bacteroids contains largely decreased levels of protein antigen groups II and III in comparison with that of free-living rhizobia (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J, J. Lugtenberg, J.Bacteriol, 171:1136-1142, 1989). Since we intend to study the molecular basis of the development of bacterium to bacteroid, we wanted to know whether these outer membrane protein differences are conserved in various plant-Rhizobium combinations, For this purpose we developed a faster assay in which cell lysates instead of isolated cell envelopes were used to analyze these outer membrane changes, With this method the previously described low levels of antigen groups II and III in isolated bacteroid cell envelopes were confirmed, Moreover the described decrease in antigen groups II and III was also found in bacteroids of Rhizobium leguminosarum by. viciae with a mutated nifA or nifK gene as well as in the non-fixing pea mutant FN1 inoculated with the wild-type strain 248, This indicates that the decrease in the antigen levels is not restricted to effective nodules, The results also showed that the decrease in antigen group II not only occurs in bacteroids from pea, but also in bacteroids from vetch, broadbean, white clover, and common bean, Antigen group III, however, remained present in bacteroids from common bean, It is concluded that the changes in antigen group II are not restricted to a specific cross-inoculation group but represent a general phenomenon in the rhizobial bacteroid differentiation process, Of the tested plants, the decrease in antigen group III was not found in bacteroids from common bean and appeared to be restricted to bacteroids from indeterminate nodules. Therefore one should expect that at least two molecular mechanisms are responsible for these outer membrane protein changes and that elucidation of these mechanisms will contribute to our understanding of bacteroid development.
    Hormonal regulation of seed development and germination in tomato : studies on abscisic acid- and gibberellin-deficient mutants
    Groot, S.P.C. - \ 1987
    Agricultural University. Promotor(en): C.M. Karssen; J. Bruinsma. - S.l. : Groot - 107
    formatie - kieming - solanum lycopersicum - plantengroeiregulatoren - kiemrust - zaadkieming - zaadzetting - zaden - tomaten - abscisinezuur - gibberellinen - mutanten - formation - germination - solanum lycopersicum - plant growth regulators - seed dormancy - seed germination - seed set - seeds - tomatoes - abscisic acid - gibberellins - mutants

    The role of endogenous gibberellins (GAs) and abscisic acid (ABA) in seed development and germination of tomato, was studied with the use of GA- and/or ABA-deficient mutants.

    GAs are indispensable for the development of fertile flowers. Fertility of GA-deficient flowers is restored by application of exogenous GAs. Fruits and seeds develop without GA, with a possible exception for the initial stage of seed growth. However, seed-produced GA delays maturation of the seeds and ripening of the fruit by one week and increases final seed and fruit weights.

    ABA levels in developing ABA-deficient mutant seeds are strongly reduced compared to wild-type seeds. Despite the strong reduction of endogenous ABA levels neither dry matter accumulation nor storage protein synthesis are affected.

    In wild-type seeds embryo-produced ABA is responsible for the development of dormancy during seed development. ABA-deficient seeds germinate viviparously in over-ripe fruits. Germination of wild-type seeds is also inhibited after harvest. Dormancy is relieved during a short period of dry storage. Stored wild-type seeds are much more sensitive to osmotic inhibition of radicle growth and germination than ABA-deficient seeds. Both types of seeds are equally sensitive to inhibition by exogenous ABA.

    GA is indispensable for tomato seed germination. GA produced by the embryo and excreted to the endosperm, induces hydrolysis of the galectomannan-rich endosperm cell walls. The hydrolysis causes weakening of the mechanical restraint of the endosperm layers that oppose the radicle tip. thereby permitting the radicle to protrude. The separation of ABA- and GA-action in time and in site is a strong argument against the existence of a hormone balance for the regulation of seed dormancy.

    Meeting of the mutation breeding contact group : Gruenbach, November 6 - 9, 1973
    Broertjes, C. - \ 1974
    Wageningen : ITAL (External report / Instituut voor Toepassing van Atoomenergie in de Landbouw no. 16) - 122
    mutanten - genotypen - plantenveredeling - gewassen - nederland - mutants - genotypes - plant breeding - crops - netherlands
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