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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.
Pflanzliche P450-Enzyme besitzen sowohl Aufgaben im Primär- und Sekundärstoffwechsel der Pflanzen als auch in der Metabolisierung von Xenobiotika einschließlich Herbiziden. Da z.B. Mais eine natürliche Resistenz gegenüber dem Triazin-Herbizid Atrazin aufweist, konnten suszeptible Wildpflanzen, die bei Feldanbau neben den Kulturpflanzen aufkommen, durch Anwendung des Herbizids ohne Schädigung der Kulturpflanzen selektiv bekämpft werden (Herbizidselektivität). Kulturpflanzen wie z.B. Tabak und Kartoffel, die keine oder nur eine unzureichende natürliche Resistenz gegenüber einem bestimmten Herbizid besitzen, können durch Agrobacterium tumefaciens-vermittelte Transformation mit einem Säuger-P450-Isoenzym (z.B. CYP1A1 oder CYP1A2) Herbizid-resistent werden. Seit einigen Jahren gibt es in dieser Richtung Bestrebungen, P450-transgene Pflanzen herzustellen. Aufgrund der überlappenden, breiten Substratspezifität des jeweils eingebrachten Säuger-P450-Isoenzyms (Ratte, Mensch) wird in den transgenen Pflanzen meist eine multiple Resistenz gegen verschiedene Herbizide mit unterschiedlichen Strukturen und Wirkmechanismen beobachtet. Vor der Vermarktung von transgenen Pflanzen müssen diese in Feldversuchen getestet werden. Dabei wird die Verträglichkeit des Genproduktes, die Eigenschaften der modifizierten Pflanze, die Expressionsstabilität des eingebrachten Fremd-Gens und mögliche ökologische Auswirkungen untersucht. Zusätzlich sollte neben der Substratspezifität des fremden P450-Isoenzyms gegenüber Xenobiotika getestet werden, ob pflanzliche Sekundärmetaboliten als Substrate in Frage kommen. Außerdem sind mögliche Einflüsse auf den normalen Stoffwechsel der Pflanzen von Interesse, die sich auf den Phänotyp der Pflanzen auswirken können. Z.B. wurde bei Cyp2c14-transformierten Tabak-Pflanzen (aus Kaninchen) eine verstärkte Seneszenz beschrieben, die sich in einem verringertem Chlorophyll-Gehalt, einem erhöhten Gehalt an Abbauprodukten der Lipid-Peroxidation und einem Abbauprodukt des Nornicotins und in einer Abnahme des Nicotin-Gehaltes äußerte. Außerdem wuchsen die Pflanzen langsamer und brauchten mehr Zeit zur Bewurzelung. Dies sind Anzeichen dafür, dass das Einbringen eines Fremd-P450-Gens in Tabak über die oxidative Veränderung der Membranlipide oder -sterole und damit über die Veränderung der Membranstruktur, durch einen hormonellen Eingriff durch Umsetzung eines Brassinosteroids oder die Unterdrückung endogener P450-Gene möglicherweise schwerwiegende metabolische Auswirkungen zur Folge haben kann. Vor diesem Hintergrund wurde untersucht, ob die Agrobakterien-vermittelte Transformation von Tabak mit der cDNA des humanen CYP1A2 Auswirkungen auf den endogenen Nicotin-Gehalt der Pflanzen zur Folge haben. CYP1A2 gehört dabei neben anderen Isoenzymen im Gegensatz zu den Hauptenzymen CYP2A6, CYP2B6 und CYP2D6 zu den Isoenzymen, die Nicotin nur bei hoher Substratkonzentration umsetzen. Nicotin besitzt dabei als natürliches Insektizid eine wichtige ökol u.s.w.
Transgenes inserted into crop plants could migrate into the genetic material of closely related wild types and cause undesirable effects - such as the development of resistance to herbicides. Background Goatgrasses (Aegilops) are genetically closely related to wheat and are often found in wheat fields, where they can be very aggressive weeds. If genetically modified wheat - for example a variety resistant to a certain herbicide - was brought onto the market on a large scale, there would be a danger of the modified genes migrating by means of wheat pollen into the genetic material (genome) of goatgrasses, making these weeds resistant to herbicides too. This risk has been demonstrated on many occasions. However, little is known about the actual probability of this gene migration occurring. Objectives The project aims to quantify to which extent the genes of genetically unmodified wheat have already mingled naturally with the genome of goatgrasses growing in the vicinity. The project also aims to assess the extent to which modified genes from transgenic wheat could spread into other, related wild types if they were to cross. Methods Various goat grasses from the Mediterranean region and North America will be investigated using genetic markers to establish how many genes have already migrated from unmodified wheat into the genome of these grasses through foreign pollination. The way these migrated genes are passed on to other related wild species will be investigated by cross-breeding various goatgrasses under both natural and experimental conditions. Significance The frequency with which wheat genes are transferred to closely related wild types and an understanding of the mechanisms by which the transferred genes spread among the wild types are important in assessing the risk associated with the development of marketable transgenic wheat varieties. In addition, the goatgrasses that will be studied are currently native predominantly around the Mediterranean and in North America but are likely to become more common in our country in the future, not least because of their migratory potential and the effects of global warming.
Establishment of modified genes If genes from genetically modified plants were to be passed on to their wild relatives, there could be serious ecological consequences, particularly if these genes were to become established in the genomes of wild plants. So far, little is known about these processes. Background One possible risk associated with genetically modified crop plants is the propagation of their genes through cross-breeding with closely related species. Scientists are seeking to assess whether resistance could be transferred from crop plants to weeds in this way and subsequently propagated in the weeds. If this was the case, these weeds would also become resistant to diseases or herbicides, an undesirable side effect. Objectives Hybrids of transgenic wheat and a close relative, jointed goatgrass (Aegilops cylindrica), will be grown under greenhouse conditions to generate information on the propagation of modified genes and whether they can become established in the genome of a wild species over several generations. The ecological consequences of this type of gene transfer will be investigated as part of the field trial with transgenic wheat (cf. Keller project I). Methods A first generation of transgenic wheat and jointed goatgrass hybrids will be bred in a greenhouse trial. Subsequent generations will be studied to see how the transgenic sequences from the wheat are passed on in the hybrids and how active they are. In addition, the ecological consequences will be assessed in a field trial. Significance There is already considerable information about the risk of cross-breeding between transgenic crop plants and their wild relatives, but little is known about the ecological consequences. The project closes this gap by investigating how the modified genes from transgenic wheat can be inherited in cross-breeding with goatgrass and whether the resulting plants have new ecological traits such as undesirable resistance.
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 Rahmen eines Vorlaeuferprojektes (TMWFK FKZ: B305-95001) wurde gezeigt, dass unter den Bedingungen des Bienendarms fremde DNA von Endosymbionten aufgenommen werden kann. Fragmente des PAT-Gens konnten auch nach laengerer Passage ausserhalb des Bienendarms nachgewiesen werden. Im vorliegenden Projekt soll geklaert werden, ob tatsaechlich eine Kopie des Transgens in den untersuchten Mikroorganismen vorlag. Dazu werden weiterfuehrende Untersuchungen an den Proben durchgefuehrt. Durch Sequenzanalyse der PCR-Fragmente soll zunaechst abgesichert werden, in welcher Form die aufgenommenen DNA-Fragmente im Genom der Mikroorganismen vorliegen. Parallel zur Analyse auf die Uebertragung des PAT-Gens soll auch die Moeglichkeit der Uebertragung eines natuerlichen pflanzlichen Gens als Bezug untersucht werden. Diese Arbeiten werden durch die Analyse von Endosymbiontenproben aus Langzeitversuchen ergaenzt, in denen gezeigt werden soll, ob eine laengere Exposition mit transgenen Pollen zu einem Anstieg der Genuebertragung fuehrt.
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.
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.
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.
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