The primary objective of this project is to examine the regulation of symbiotic plant genes induced during the nodulation of alfalfa. The proposed program focuses on a collection of alfalfa genes whose expression requires the invasion and which have been previously cloned. These genes are probably involved in functional activities associated with ammonia production and are good candidates for direct control by developing bacteroids. The study of symbiotic nitrogen-fixation is significant because the nodulation process is developmentally regulated and can address fundamental questions about cell growth and differentiation. Nodulation also provides a system to examine the coordinated expression of both bacterial and plant genes in a symbiotic state. Finally, the ability of legumes to symbiotically fix nitrogen has become an important factor in maintaining soil productivity and decreasing the fertilizer requirements for other crops, especially in developing countries. The overall strategic approach will be to use a combination of antibody and recombinant DNA techniques to probe the structure of these symbiotic genes and examine their transcriptional and translational properties. Specifically, antibodies directed against each of the gene products will be isolated from animals immunized with a B-galactosidase fusion protein product produced in lambda gtll recombinants. Using these specific antibodies, we will examine a developmental time course of gene expression and identify the sub-cellular localization of each specific protein in both wild-type and mutant nodules. Standard sequencing technology will be used to sequence both genomic and cDNA clones. This will describe coding region of the gene and the resulting amino acid sequence. The promoter regions for each of the symbiotic plant genes will be identified by S1 mapping of the region 5' to the cDNA. Alfalfa has a good tissue culture system, can be regenerated from single cells, and can be transformed to produce transgenic plants. This project will generate new information about symbiotic plant genes and provide the necessary tools (antibodies, promoters) to further study gene regulation and function, taking advantage of alfalfa's unique biological properties.
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