The long term goal of this project is to understand the molecular basis of the developmental events responsible for nodulation of plants by rhizobia. The Rhizobium meliloti/alfalfa symbiosis is a particularly attractive model system for the study of developmental issues since it is now possible to genetically manipulate R. meliloti with a resolution and ease comparable to that of manipulations of E. coli. In our previous work, we have shown the R. meliloti strain Rm 1021 synthesizes a complex acidic exopolysaccharide that is required for nodule invasion and nodule development. Our studies offered insights into the symbiotic importance of certain structural features of this exopolysaccharide and into the relationship of its regulation to its symbiotic role. We also showed that R. meliloti has a cryptic capacity to synthesize a second exopolysaccharide of novel structure that can substitute for the role(s) of the first exopolysaccharide in nodulation. We will continue our genetic analyses of exopolysaccharide production by R. meliloti focusing on such issues as the roles of the acetyl and pyruvyl modifications of these exopolysaccharides and on the regulation of their synthesis during the nodulation process. We will use a cell-free system and our extensive collection of mutants to carry out biochemical analyses of he synthesis of these two R. meliloti exopolysaccharides. We will analyze the possible roles of R. meliloti oligosaccharides to function as signals to the plant during nodulation and will determine the structural features of the oligosaccharides necessary for this role. We will test other hypotheses we have developed concerning the roles of exopolysaccharides in nodulation such as helping the plant to evade plant defense responses, binding extracellular factors or enzymes, or playing physical roles in nodulation such as being involved in cap formation on root hairs or serving as the matrix in infection threads. We will also explore other aspects of polysaccharide involvement in nodulation such as whether R. meliloti synthesizes other polysaccharides such as the cellulose or the gel-forming polysaccharide curdlan and, if so, whether these are necessary for normal nodulation to occur. The proposed studies should offer insights into molecular strategies underlying developmental changes, into the roles of exopolysaccharides in host-pathogen interactions, into the biosynthesis of such polysaccharides, and into the function of oligosaccharides as intercellular signals.
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