The biotrophic fungus Ustilago maydis infects corn and induces the formation of tumors. In order for the fungus to proliferate in the infected tissue, U. maydis has to redirect the metabolism of the host to the site of infection. We wish to elucidate how this is accomplished. To this end we will perform transcript profiling during the time course of infection for both, the fungus and the maize plant. This will be complemented by metabolome analysis of different tissues during infection as well as by apoplastic fluid analysis. The goals will be to identify the carbon sources taken up by the fungus during biotrophic growth, to identify the transporters required for uptake, determine their specificity and elucidate how these carbon sources are provided by the plant. Fungal mutants affected in discrete stages of pathogenic development will be included in these studies. Likely candidate genes for carbon uptake/supply as well as for redirecting host metabolism will be functionally characterized by generating knockouts in the fungus and by isolating plants carrying mutations in respective genes or by generating transgenic plants expressing RNAi constructs.
This project is aimed at the characterization of the systemic reprogramming in barley, which modulates the compatible interaction with the biotrophic leaf pathogen Blumeria graminis f.sp. hordei upon root infestation with the mutualistic endophyte Piriformospora indica. We have recently shown that the basidiomycete P. indica - upon successful establishment in the roots - reprograms barley to salt stress tolerance, resistance to root diseases and higher yield (Waller et al., 2005). Successful powdery mildew infections in barley leaves are also disturbed by the mutualistic fungus. These processes are associated with a strong change in plant metabolism, especially with a drastic alteration of leaf and root antioxidants. On the basis of these findings we will perform an in-depth analysis of the barley metabolome (B6) and transcriptome (B7) with two specific foci: First, to elucidate the process of establishment of the mutualistic fungus within the barley roots; second, to characterize elements of the systemic response in leaves leading to an interruption or failure of compatibility processes required for successful establishment of biotrophic leaf pathogens like Blumeria. New gene candidates will be pre-selected systematically for their regulatory role in compatibility by means of transiently transformed barley leaves upon Blumeria inoculation. Stable transgenic barley and maize lines (B3) generated with verified gene candidates and genes identified by other projects (A1, A2, B5, B6) will be tested with Blumeria and P. indica. By comparing candidate genes in the different plant - microbe systems, we will identify common regulatory processes, metabolites and metabolic networks implicated in compatibility including those required for successful interactions with mutualistic fungi.
Does fungal resistance in transgenic wheat harm beneficials? Some fungi cause devastating diseases; others are beneficial to the plant, facilitating its uptake of nutrients. If plants are genetically engineered to make them resistant to fungal diseases, this resistance could also have a negative impact on so-called beneficials. Background Mildew and other fungal diseases have to be controlled in agriculture with fungicides that harm the environment. To reduce the use of fungicides, plant breeders are attempting to modify the genetic material of crop plants to enhance their resistance to fungi. However, many plants naturally form close relationships (symbioses) with beneficial fungi (mycorrhizas) that play a major role in enabling the plant to absorb minerals such as phosphorus and nitrogen from the soil. Laboratory trials have shown that enhanced resistance to fungi in genetically modified crops can have a negative effect on symbioses with beneficial fungi. Objectives A major field trial with transgenic wheat (cf. Keller project I) will investigate whether these laboratory results can be extrapolated to conditions in the open. The project will study the effect of enhanced resistance to fungi on the colonization, function and diversity of special symbiotic fungi that are found in the roots of wheat plants. Methods The planned trials will use both conventional microscopy and new genetic methods. The colonization of the plants' roots by symbiotic fungi will be determined by identifying the fungal spores under the microscope and by quantifying the fungal DNA. Trials with special nutrient capsules will provide information on the extent to which the function of these fungi changes. The spores will also be studied to determine the diversity of fungi in the plants' roots. Significance In the struggle to achieve sustainable agriculture and promote healthy soils, it is important to accurately assess the extent to which fungal resistance in crop plants can be reconciled with the symbiotic fungi living in their roots. The project will develop a basis for this work.
Do genetically modified strawberries pose a threat to wild varieties? Do genetically modified strawberries pose a threat to wild varieties? Strawberries are an important niche product in Switzerland. Breeders are experimenting with genetic engineering methods to enhance the marketability of this product. There are risks inherent in this approach since the transfer of modified genes to wild strawberries could endanger the continued existence of the wild varieties. Background Transgenic varieties of strawberry with higher yields and enhanced root development already exist. The first release trials have already begun in Italy. However, if transgenic varieties of strawberry cross-breed with wild ones, there may be negative effects. The hybrids produced in this way are often sterile, yet by back-crossing with wild types or by producing prolific numbers of offshoots they can penetrate the native flora and displace it. Objectives This project has two basic goals. First, it seeks to assess the extent to which transgenic strawberries are capable of cross-breeding with their wild relatives. Second, it seeks to investigate the possible ecological impact of such crossbreeding under various environmental conditions in order to assess the risks associated with cultivating transgenic strawberries in the open. Methods Greenhouse experiments with honey bees, the most important pollinators of strawberries, will be carried out to show whether and how efficiently natural pollination occurs between transgenic and wild strawberries. Genetic methods will be used to determine how frequently foreign pollination between cultivated and wild strawberries has already occurred in the open in the past. The possible ecological implications of this will be quantified using life history data such as growth and competitive pressure. In these experiments, transgenic and artificially cross-bred strawberries from the laboratory will be planted in various soils. Significance The cultivation of transgenic plants is associated with potential risks for their wild relatives. Scientists have warned that the latter could become extinct as a result of undesirable cross-breeding. However, to date the true extent of these risks has barely been investigated. This project aims to close this gap by generating basic data with transgenic and wild strawberries as model organisms. These data could ultimately be relevant for other related crop plants such as apple trees or cherry trees.
Im Projekt soll die ökologische Relevanz eines horizontalen Gentransfers innerhalb der endophytischen Mikroflora von Forstgehölzen untersucht und bewertet werden. Hierzu wird in einem Modellsystem mit Pappel-Stecklingen/Jungpflanzen ein Gentransfer über die bakterielle Konjugation in die Endophytenmikroflora induziert. Der Gentransfer erfolgt durch ein rekombinantes Plasmid mit einem geeigneten Reportergen, das in vitro in einen Agrobakterien- und einen endophytischen Bakterienstamm eingebracht wurde. Es wird geprüft, inwieweit sich das rekombinante Plasmid in der Endophytenmikroflora etablieren kann. Ein weiterer Schwerpunkt ist die Abschätzung des Entlassens des rekombinanten Plasmids aus der Endophytenmikroflora der Pappel in verschiedene Umweltmedien (Boden und zersetztes Pflanzenmaterial).
Phosphinothricin (PPT, Wirkstoffname: D,L-Glufosinate-Ammonium, Praeparatename: BASTA bzw. LIBERTY bei der Anwendung in transgenen Pflanzen) ist ein nicht selektives Herbizid, welches bei der Anwendung in gentechnisch unveraenderten Pflanzen nicht in Kontakt mit der Kulturpflanze kommt und damit dort zu keinen Rueckstaenden fuehrt. Beim Abbau transgener, PPT-vertraeglicher Kulturen wirkt PPT selektiv gegen die Unkraeuter. Durch die dann moegliche Nachauflaufanwendung (Benetzung) kommt die PPT-resistente Kulturpflanze mit der vollen Aufwandmenge und damit mit wesentlich hoeheren Wirkstoffmengen in Beruehrung. Veroeffentlichte Untersuchungen zum Metabolismus von PPT liegen nur in geringem Umfang fuer PPT-empfindliche pflanzliche Zellkulturen vor und beinhalten einen stufenweisen, an der Aminogruppe angreifenden Abbau der Substanz. In transgenen Pflanzen ist diese Aminogruppe des Herbizidmolekuels durch Bildung des Acetylphosphinothricins blockiert. Ziel der Untersuchungen ist zu pruefen, ob dieser Metabolit nur in resistenten Pflanzen auftritt, weiter abbaubar ist, und ob in resistenten Pflanzen im Vergleich zu den sensitiven mit hoeheren Rueckstandsmengen und anderen Metaboliten zu rechnen ist. Gleichzeitig soll untersucht werden, ob im Abbauverhalten Unterschiede zwischen der D- und der L-Form des Herbizids bestehen. Durch Aufklaerung der Abbauwege und Abbauprodukte sowie Bilanzierung der Rueckstandsmengen in verschiedenen Pflanzenteilen sollen Basisdaten ueber Persistenz, Abbauverhalten und Verteilung zur Sicherheitsbewertung von PPT-Rueckstaenden in transgenen Nahrungspflanzen erarbeitet werden.
Gentechnisch veraenderte Pflanzen enthalten haeufig als Selektionsmarke das Antibiotika-Resistenz-Gen Neomycin-Phospho-Transferase (NPTII) in ihrem Erbgut. Die Kultivierung solcher Pflanzen im Freiland wirft die Frage auf, ob das NPTII-Gen wieder in Bakterien zurueckgelangen kann. Bei den zu unterscheidenden Pflanzen handelt es sich um Tabak oder Petunien, die mit unterschiedlich modifizierten NPTII-Genen transformiert worden sind. Es soll ueberprueft werden, ob durch die Verrottung dieser Pflanzen das NPTII-Gen in die Erde gelangen und dort von Bodenbakterien aufgenommen und weitervererbt werden kann. Zum Nachweis eines solchen etwaigen horizontalen Gentransfers wird eine hochempfindliche und spezifische Nachweismethode ('Polymerase-Chain-Reaction') eingesetzt. Die Untersuchungen zum Gentransfer werden sowohl im Gewaechshaus (Tabak und Petunie) als auch im Freiland (Petunie) durchgefuehrt. Dabei sollen Erkenntnisse gesammelt werden, inwieweit Gentransferereignisse im Freiland durch analoge Versuche im Gewaechshaus vorhergesagt werden koennen.
SUNBIOPATH - towards a better sunlight to biomass conversion efficiency in microalgae - is an integrated program of research aimed at improving biomass yields and valorisation of biomass for two Chlorophycean photosynthetic microalgae, Chlamydomonas reinhardtii and Dunaliella salina. Biomass yields will be improved at the level of primary processes that occur in the chloroplasts (photochemistry and sunlight capture by the light harvesting complexes) and in the cell (biochemical pathways and signalling mechanisms that influence ATP synthesis). Optimal growth of the engineered microalgae will be determined in photobioreactors, and biomass yields will be tested using a scale up approach in photobioreactors of different sizes (up to 250 L), some of which being designed and built during SUNBIOPATH. Biomethane production will be evaluated. Compared to other biofuels, biomethane is attractive because the yield of biomass to fuel conversion is higher. Valorisation of biomass will also be achieved through the production of biologicals. Significant progress has been made in the development of chloroplast genetic engineering in microalgae such as Chlamydomonas, however the commercial exploitation of this technology still requires additional research. SUNBIOPATH will address the problem of maximising transgenic expression in the chloroplast and will develop a robust system for chloroplast metabolic engineering by developing methodologies such as inducible expression and trans-operon expression. A techno economic analysis will be made to evaluate the feasibility of using these algae for the purposes proposed (biologicals production in the chloroplast and/or biomethane production) taking into account their role in CO2 mitigation.
Based on the Ac/Ds two element transposition system from maize an activation tagging approach is suggested for the hybrid aspen (Populus tremula x tremuloides) line -Esch5-. The proposed approach is based on results obtained from our earlier work on the genetic transfer of the maize transposable element Ac and its functional analysis in hybrid and pure aspen lines. It was shown that the Ac element is active in aspen and reintegrates elsewhere in genomic regions in high frequency. However, a two element transposon tagging system where Ac and Ds are put together in crosses is not feasible in trees due to the in part long vegetative phases. To overcome this barrier, an inducible two element Ac/ATDs element system is suggested to induce activation tagged variants following two independent transformation steps. In combination with a 35S enhancer tetramer and outward facing two CaMV 35S promoter located near both ends of the ATDs element, expression of genes can be elevated which are located adjacent to the new integration site of the element. As selective marker for ATDs transposition, both knocking-out the expression of a phenotypic marker (rolC gene) and a negative selection marker gene (tms) are considered. Thus, the transposition can easily be screened in primary transgenic lines.
How does fungal resistance of transgenic wheat behave in the open? Fungi, and most particularly mildew, cause enormous losses in wheat harvests. To overcome this, wheat was genetically engineered to resist mildew. But there is still very little information about how this resistance functions in open cultivation. Background Mildew and other fungi cause tremendous damage in wheat production, necessitating the use of sprayed crop-protection products. It has been possible to use genetic engineering to overcome this problem by incorporating a specific barley gene in the wheat genome. This gene produces proteins that degrade the cell walls of fungi and destroy the pests. Little is known, though, about the efficacy of this method in open cultivation or the conceivable risks. Objectives The project aims to investigate how fungal resistance in genetically modified wheat behaves in the open. The aim is to measure the efficacy of this resistance against fungal diseases and to assess the potential benefit for agriculture. Methods The efficacy of mildew resistance will be investigated in three successive years as part of the field trial with transgenic wheat (cf. Keller project I). Among other things, the activity of the resistance genes and the productivity of the wheat lines will be measured. Parallel trials will check the results of the field trial under greenhouse conditions. Significance Plants behave differently in the greenhouse and in the open. It is therefore necessary to test the action of the additional resistance genes in field trials. This project will evaluate both resistance to true mildew and resistance to other pathogenic fungi.
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