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.
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.
What impact does transgenic maize have on soil fertility? Among the factors that determine soil fertility is the diversity of the bacteria living in it. This is in turn affected by the form of agriculture practiced on the land. What role do transgenic plants play in this interaction? Background Soil fertility is the product of the interactions between the parental geological material from which the soil originated, the climate and colonization by soil organisms. Soil organisms and their diversity play a major role in soil fertility, and these factors can be affected by the way the soil is managed. The type of farming, i.e. how fertilizers and pesticides are used, has a major impact on the fertility of the soil. It is known that the complex interaction of bacterial diversity and other soil properties regulates the efficacy of plant resistance. But little is known about how transgenic plants affect soil fertility. Objectives The project will investigate selected soil processes as indicators for how transgenic maize may possibly alter soil fertility. The intention is in particular to establish whether the soil is better able to cope with such effects if it contains a great diversity of soil bacteria. Methods Transgenic maize will be planted in climate chambers containing soils managed in different ways. The soil needed for these trials originates from open field trials that have been used for decades to compare various forms of organic and conventional farming. These soils differ, for example, in the way they have been treated with pesticides and fertilizers and thus also with respect to their diversity of bacteria. The trial with transgenic maize will measure various parameters: the number of soil bacteria and the diversity of their species, the quantity of a small number of selected nutrients and the decomposition of harvest residues. It will be possible to conclude from this work how transgenic plants affect soil fertility. Significance The project will create an important basis for developing risk assessments that incorporate the effects of transgenic plants on soil fertility.
How do insecticidal proteins from transgenic plants behave in soil? Many transgenic plants produce proteins that kill certain insect pests that feed on the plants. When these plants are cultivated, proteins of this type also pass into the soil and may harm other organisms there. Background An increasing number of transgenic plants are being cultivated worldwide that produce insecticidal proteins, known as Cry proteins, to defend themselves against insect pests. When these plants are grown, some of the Cry proteins pass into the soil either with dead plant material or directly via the plants' roots. It cannot be ruled out that these proteins may have negative effects on the soil. There are concerns, for example, that the Cry proteins damage beneficial soil organisms and bacteria, and that insect pests could become resistant to these proteins. The possible extent of these effects depends on how strongly Cry proteins adhere to solid components of the soil. Objectives The project aims to achieve a precise understanding of the way Cry proteins adhere to various components of soil. This knowledge will allow assessing the stability of these proteins in soils, the distances over which the proteins are transported in soil and the extent to which beneficial soil organisms come into contact with them. Methods Cry proteins adhesion will be studied to different soil components - including quartz sand, clay minerals and humus - and three selected soils encountered in Swiss agriculture. The adhesion of Cry proteins will be examined directly on the surfaces of the soil components, i.e. on a microscopic scale, using specific instruments. The results will form the basis for the development of a computer model to predict the adhesion and transportation Cry proteins in various soils. Significance The risk of possible damage being caused by Cry proteins in soils can only be assessed if the strength of the adhesion of these proteins to soil components is known. The experimental studies needed to provide this information will be carried out in this project. In addition, the results will be incorporated into a model that will allow estimating possible negative effects of Cry proteins in various agricultural soils.
How digestible is transgenic wheat for earthworms? Genetically modified crops are intended to be toxic for the pests that attack them. At the same time, however, they could harm beneficial organisms. Background Crop plants can be genetically modified to make them immune to pathogens such as fungi, or unpalatable or toxic for pests that feed on them. The overriding objective of plant breeders is to reduce the use of crop protection products. The same substances may, however, be harmful to animals that are important for plants, such as woodlice and worms, as they play a central role in decomposing plant material and releasing nutrients into the soil. Objectives The diversity of species and activity of selected soil-dwelling organisms are expected to provide information on the impact of transgenic plants on these important groups of animals. In addition, nutrient uptake and reproduction of selected soil-dwelling organisms will be compared in areas used to grow genetically modified wheat and areas used to grow conventional wheat. Methods Arthropods (such as woodlice) and segmented worms (such as earthworms) are beneficial invertebrates that live in the soil. Their diversity will be investigated using soil samples as part of the field trial with transgenic wheat (cf. Keller project I). Their activity and nutrient uptake will be determined by burying a constant volume of leaf material derived from transgenic wheat plants and conventional wheat plants for a period of several months. The amount eaten by the soil-living organisms will subsequently be measured. Significance Little is known about the effect of substances that may be released into the soil from the transgenic plants being investigated here. The project is using arthropods and annelid worms as an example of how to investigate this question. The ecologically oriented design of the project will also create a basis for assessing the risk of transgenic plants affecting soil fertility in open cultivation.
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.
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.
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