ENOD40 encodes a peptide growth factor

Rhizobium bacteria induce the formation of nodules on the roots of leguminous plants. The nodules create the right biological niche for the rhizobia to carry out biological nitrogen fixation by which atmospheric nitrogen is reduced to ammonia. The nodule is a new organ that provides the plant with a nitrogen source for its growth and development. The formation of a nitrogen fixing root nodule is the final result of an extensive collaboration between the plant and the bacterium, which starts with the exchange of signals. The plant roots secrete flavonoids, which attract rhizobia and induce the expression of nodulation (nod) genes in the rhizobia. Due to the nod gene expression, specific lipochitooligosaccharide signals are produced, the so-called Nod factors, that induce several responses in the roots as a result of which nodule formation can start. The first plant responses are root hair deformation, expression of several plant genes and the mitotic reactivation of root cortical cells which leads to the formation of a nodule primordium. In chapter I a general overview is given of the signal exchange leading to the formation of a functional root nodule.The aim of the research, described in this thesis, was to analyse the role of the early nodulin gene ENOD40 in nodule development. To adress this issue, ENOD40 expression was determined in nodules and its actvity was studied in an in vitro model system. First an ENOD40 clone was isolated from pea using the available soybean ENOD40 cDNA as a probe. This made it possible to compare the expression pattern of ENOD40 in a determinate (soybean) and indeterminate (pea) nodule by in situ hybridisation. using the soybean and pea ENOD40 clones, respectively, as probes (chapter 2). Chapter 3 describes the isolation and characterisation of the soybean ENOD40-2 gene. A transcriptional fusion between the ENOD40-2 promoter and the β-glucuronidase reporter gene was used in Agrobacterium rhizogenes mediated transformation of Vicia hirsuta. Root nodules were induced on the transgenic hairy roots by infection with Rhizobium leguminosarum bv. viciae and activity of the ENOD40 promoter was analysed using GUS assay.Expression of the ENOD40 gene is detectable early after infection in the pericycle of the root, before cortical cell divisions take place. It was assumed that ENOD40 expression might be required for the induction of cortical cell division, and might function by influencing auxin and/or cytokinin levels which play a role in the induction of cell division. This hypothesis was tested in a model system, the tobacco protoplast cell division assay. With this assay, the interaction of ENOD40 with auxin and cytokinin was studied, and ENOD40 was shown to induce tolerance of high auxin and cytokinin concentrations in tobacco protoplasts (chapter 4, 6). Using the tobacco protoplast cell division assay it was demonstrated that an oligopeptide of 10 to 13 amino acids encoded by ENOD40 is the compound responsible for this effect. In addition, a conserved region in the 3' UTR of ENOD40 also has an effect (chapter 4).Tobacco cells are able to respond to a soybean ENOD40 cDNA clone and to the soybean ENOD40 peptide. This indicates in tobacco homologous genes might be present. The cloning of these genes by PCR based methods is described in chapter 4 and 5. The presence and activity of ENOD40 in legumes and a non legume indicates ENOD40 might play a general role in plant development. Therefore, in the concluding remarks (chapter 7) it is discussed whether peptides can play a more common role in plant development and whether and how the 3' UTR of ENOD40 mRNA might function.