Determination of the traits that determine competitive success in root colonisation and nodulation by Rhizobium leguminosarum

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The rhizobia are one of the best-characterised groups of plant-associated bacteria that must be able to colonise the roots of legumes before they can initiate a successful infection process, resulting in nodulation and nitrogen fixation. Colonisation of the rhizosphere by microorganisms in general is one of the primary determinant of plant productivity, yet is very poorly understood. Likewise, competition for nodulation success is poorly understood, even though legumes account for a substantial fraction of the biosphere¿s fixed nitrogen. At a practical level the use of agronomically desirable rhizobia is often limited by their lack of colonisation and nodulation competitiveness. This lack of understanding of colonisation and nodulation competitiveness is largely because it is a complex process involving multiple biotic and abiotic factors that have been difficult to examine experimentally. However, new genome based technologies such as signature tagged mutagenesis (STM), microarray analysis and biosensors allow a systematic approach to be adopted to the study of this problem. STM is particularly powerful because it allows pools of individually tagged mutants to be competed against one another and the successful strains identified by subsequent array hybridisation against the tags spotted on glass slides. Since I have developed colonisation and nodulation competition assays for R. leguminosarum strain 3841, am developing whole genome microarrays for this organism and have developed high-throughout Gfp biosensors I am well placed to use these new strategies to tackle the genuinely important problem of colonisation and nodulation competitiveness. I therefore propose to use STM first to isolate mutants of Rhizobium leguminosarum strain 3841 essential for colonisation (col) and nodulation competitiveness (noc). By starting with mutants with a clearly defined phenotype it avoids the problem of producing large amounts of transcriptome proteome data lists with no function. Next microarray analysis will be used to examine the global expression of genes in the pea rhizosphere but with a sharp focus on the expression of the col and noc genes. This will allow the examination of the environmental regulation of these genes and permit the rational design of Gfp biosensors focused on those col and noc genes that are induced in crucial microenvironments, such as root hairs and nodule infection threads. These genes have been among the most difficult to investigate, in spite of them being vitally important for root colonisation and nodulation. Gfp biosensors will allow precise spatial and temporal mapping of key col and noc gene expression, enabling their sites of action to be pinpointed. Comparative array analysis of the expression R. leguminosarum genes in the pea, alfalfa and sugarbeet rhizosphere will also be carried out to determine if there are common or discrete sets of genes induced in the rhizospheres of these plants. The induced genes will be compared to the genomes of other plant-associated bacteria to look for common colonisation genes.