Ethan Royal Signer - US grants

The long term goal of this research is to understand the molecular mechanism of the complex developmental process by which Rhizobium meliloti forms symbiotic nitrogen-fixing nodules on the roots of its host alfalfa. Genetic and molecular biological strategies commonly used with Escherichia coli, lately made possible in this system, will be combined with biochemical, immunological and cell physiological techniques in a multidisciplinary approach based on the complementary expertise of two established laboratories. As specific aims, genetic tools will continue to be developed, including transposon Tn5 derivatives for making operon fusions in vivo, Hfr-like strains for conjugal mapping, "maxicells" for visualizing proteins, and host-range and conditional phage mutants for improved transduction. Genetic analysis of exopolysaccharide (EPS) required for effective nodulation will also continue, including cloning and characterization of genes, isolation of new mutants and identification of additional loci. Both extracellular products and lipid-bound biosynthetic intermediates of EPS-deficient mutants will be characterized biochemically. Morphological studies will further characterize the atypical "empty" nodules of EPS mutants, the interaction with plant roots of mutants having altered EPS, and, by immunofluoresence with monoclonal antibodies, the distribution of EPS in nodule tissue. The ability of isolated EPS to restore effective fixation to mutants will be analyzed further. Regulation of symbiotic genes by plant products will be approached by fusion of nodulation operons to easily assayed reporter genes. This system will serve as a model for the developmental interaction of prokaryotic and eukaryotic cells, of which there are many examples in the area of human health. More specifically, this work will help elucidate the mechanism of biological nitrogen fixation in legumes, with immediate relevance to the health-related issues of plant productivity, agronomic energy conservation and provision of nitrogen in the diet.

Human health depends strongly on adequate nutrition. Symbiotic fixation of atmospheric dinitrogen by legume rhizobia is the major source of nitrogen in the human diet, as well as the major factor limiting legume crop yield. This project aims to characterize the alterations entailed in differentiation of the alfalfa symbiont Rhizobium meliloti from free-lving bacteria, which do not fix nitrogen, to the nitrogen-fixing bacteroid form. Three interrelated studies will be carried out. (1) Fix genes, required for differentiation, will be identified by transposition mutagenesis with a Tn5-lac promoter probe, which will allow characterization of regulatory circuitry. (2) Changes in the outer and inner bacterial membranes will be characterized as a function of bacterioid differentiation. (3) The heat shock response, which depends on a specific RNA polymerase sigma subunit, will be characterized in R. meliloti, as a possible genetic approach to identification of their sigma subunits suspected to be involved in differentiation. These studies should begin to define the molecular changes required for differentiation of the bacteria to a form that can fix nitrogen. Such information will ultimately be necessary for genetic engineering of nitrogen fixation, with benefits to human health and agronomy.

The ultimate goal of this project is to establish how the alfalfa symbiont Rhizobium meliloti crosses the plant cell walls during establishment of nitrogen fixing nodules. Results of others suggest this involves degradation of cell wall polysaccharides (cellulose, hemicelluloses and pectic substances). Preliminary results have identified an activity that releases reducing sugars from polysaccharides as well as an activity (probably different from the preceding one) that degrades polysaccharide in an agar plate test. In addition, DNA sequences may have been identified that hybridize with a cloned pectate lyase gene from Erwinia chrysanthemic. These results will be pursued to characterize the genes and gene products involved. This should elucidate the mechanism of rhizobial invasion of legume roots, which in turn should provide valuable information for genetic engineering of nitrogen fixation, with corresponding benefits to human health and nutrition.

The long-term objective of this project is to characterize homologous genetic recombination in plants. This work will focus primarily on the small crucifer Arabidopsis thaliana as a model system, and secondarily on the related brassica cauliflower (Brassica oleracea var. botrytis. Specific aims are: (1) to complete purification of recominase activity and characterize the enzyme(s); (2) to clone and sequence the corresponding gene(s) and flanking regulatory regions; (3) to construct Arabidopsis lines carrying markers suitable for scoring various crossover events, and begin characterizing those events in various conditions. Future likely directions include determination of recombinase genetic regulatory mechanisms, of the effects of altered recombinase gene expression, and of the role of mitotic and meiotic recombination in the plant life cycle. This work, besides providing information about a fundamental biological process, could eventually allow that process to be manipulated experimentally as well. In the long term that could be of value in the genetic engineering of agronomically important crops, to the benefit of human nutrition and health.

ABSTRACT: 9318929, PI-Signer: Research into plant biology is hampered by the lack of the technology necessary for site specific recombination. With that as the ultimate goal, the specific aims of this proposal are to develop in vivo genetic methodology to exploit: 1. Site specific double-strand break repair by HO endonuclease, which should be of value in gene targeting, mosaic analysis, and marker eviction. 2. Site specific genomic rearrangements by FLP recombinase, which should generate deletions, inversions and translocations. Plant molecular biology is currently limited by supporting genetic technology, particularly in the model plant Arabidopsis thaliana. This project will develop in vivo genetic methodology, initially for Arabidopsis but in forms suitable for other plant species too. Experiments are planned to exploit expression in planta of two yeast site specific enzymes, HO endonuclease and FLP recombinase. Areas to be explored are gene targeting, marker eviction, mosaic analysis, and genomic rearrangements (deletions, inversions, translocations). This technology will allow the full range of modern recombinant DNA technology to be brought to bear on plant systems, for both fundamental research and improved engineering of agronomic crop species.

This project is attempting to engineering a bacterium, using no more than currently available technology, intro an intracellular, seed-transmissible endosymbiont that functions as a plant 'organelle'. The approach is based on a screen designed to select directly for endosymbiosis. An SGER would allow us to test the feasibility of the method. We will construct Bacillus subtilis Kan (kanamycin-resistant) Hyg (hygromycin-resistant) Gus (B- glucuronidase); derive L-forms from it; introduce these into tobacco protoplasts; select for callus simultaneously resistant to Kan on the presence of endosymbiotic 'organelles'. If this proves feasible, it would permit a full-scale research project to develop conditions for optimization of selection and regeneration of endosymbiotic callus into whole plants. Several arguments suggest the approach is feasible in principle. These include not only the straightforward selection and simple technology, but also the large number of endosymbioses found in nature, and particularly the reports of laboratory-induced endosymbiosis in Amoeba and plant cells. %%% Organelle engineering would allow plants to be equipped with agronomically valuable metabolic and physiological capabilities that are too complex to be introduced by gene transfer, would also provide a practical system for system endosymbiosis in the laboratory.

Plant molecular biology is currently limited by supporting genetic technology, particularly in the model plant Arabidopsis thaliana. This project will develop in vivo genetic methodology, initially for Arabidopsis but in forms suitable for other plant species too, that exploits the analytical power of events targeted in vivo to specific loci. That will include targeted gene integration, by one or more of several novel approaches (terminal homology vectors; telomere vectors; transformation mutants; positive-negative selection) designed to be extendable to plant species hard to transform; and targeted double-strand breaks (in ectopic recombination; in transposition), to promote homologous recombination. This will allow the full range of modern recombinant DNA technology to be brought to bear on plant systems, for both fundamental research and breeding of agronomic crop species.//