Bacteria of the genus Rhizobium play a very important role in agriculture by inducing nitrogen-fixing nodules on the roots of legumes such as peas, beans, clover and alfalfa. This symbiosis can relieve the requirements for added nitrogenous fertilizer during the growth of leguminous crops. The Rhizobium group is studying the bacterial and legume genes involved in establishing and maintaining the symbiosis. This will provide background knowledge for use in applied objectives as well as yielding a wealth of fundamental knowledge with wide implications. Underpinning the work is a continuing investigation of the bacterial and plant genes specifically induced during the symbiosis. The communications that occur between the plant and the rhizobia during nodule formation and maintenance constitutes a novel opportunity to study signal transduction in a plant system. The expression of "nodulation" genes in the bacteria is activated by signals from plant roots and as a result the bacteria synthesise signals that induce a nodule meristem and enable the bacteria to enter this meristem via a plant-made infection thread. The chemical signals synhesised by the bacteria are based on a modified amino acid (homoserine lactone) carrying a variable acyl chain substituent, and are called acyl homoserine lactones (AHLs). By detecting and reacting to these chemicals, individual cells can sense how many cells surround them, and whether there are enough bacteria, i.e. a quorum, to initiate the change towards acting in a multicellular fashion. This is known as 'quorum sensing' and this laboratory is coordinating an EU Consortium on 'Rhizosphere Communication' to investigate the extent to which specific plant-growth-promoting bacteria use AHL-based quorum sensing regulation of important physiological traits, and the degree of cross-talk with plant pathogens.
Within the nodule meristem, the bacteria induce specialised genes required for nitrogen fixation. Within the nodule and infection thread, plant products including glycoproteins and glycolipids are synthesised de-novo. Monoclonal antibodies directed against such plant products have been isolated and used to analyse the spatial and temporal expression of plant-made components important in the developing nodule. Specific pea genes induced during the symbiosis have been identified and have given new insights into novel roles of various gene products expressed during the infection process that leads to nodule development. This work has implications for the improvement of symbiotic nitrogen fixation and provides an important tool in plant cell biological studies and in the field of plant-microbe interactions.
The formation of nitrogen fixing nodules on legumes requires co-ordinated expression of several bacterial and plant genes. Initial stages of nodule formation require expression of specific nodulation (nod) genes by rhizobia. The nodABCFELMN gene products are involved in the synthesis of a group of signal molecules (Nod factors) that induce nodule morphogenesis. These signals are acylated derivatives of an oligo N acetyl-glucosamine polymer (4 or 5 residues long). NodF and NodE determine the type of N-acyl group (C18:4 in Rhizobium leguminosarum biovar viciae) on the first glucosamine on the oligomer, whilst NodL carries out an O-acetylation of the first glucosamine residue. The role of these Nod factors in efficient nodulation will be analysed with regard to effects on competitive nodulation in different varieties of pea. In parallel, R. leguminosarum biovar viciae secretes a Ca2+ binding protein (NodO) that probably interacts directly with root cell membranes to stimulate the infection process. The mechanism of secretion of this protein will be analysed by identifying the genes that encode the protein translocation machinery. Subsequent stages in nodulation involve intimate contact between the Rhizobium and plant cell surfaces, and the molecular interactions that occur between them will be studied. Bacterial surface components include the lipopolysaccharide (LPS); the role of its structure in the development of normal nodules will be studied using monoclonal antibodies and bacterial mutants altered in LPS structure. Also important for symbiotic nitrogen fixation is a specialised set of cytochromes required for respiration in the low oxygen concentration found in legume nodules. The assembly and regulation of these cytochromes will be analysed with a view to improving the efficiency of nitrogen fixation. Plant-made determinants important in the symbiosis include glycoproteins and glycolipids including inositol derivatives. Using monoclonal antibodies or specific antisera, individual components have been localised to the infection thread matrix, peribacteroid space and peribacteroid membrane. Future studies will focus on the role of host defence systems in controlling the process of tissue and cell invasion by Rhizobium. In addition, the differentiation of the peribacteroid membrane will be studied as a model system for membrane biogenesis and vesicle targetting in plants. These studies will exploit the wide range of symbiotically defective mutants that are available for both the bacterial and higher plant partners. Experimental techniques will range from molecular cell biology (immunolocalisation, in situ hybridisation), through to standard molecular genetics (PCR, cDNA cloning, transformation and the use of transgenic plants). Molecular mapping of pea genes required for nodulation is underway.
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