Fine mapping of the sym2 locus of pea linkage group I.

The symbiotic interaction between Rhizobium bacteria and leguminous plants results in the formation of root nodules which are specific, nitrogen-fixing organs that supply the plant with ammonia required for its growth. The formation of a nitrogen-fixing root nodule involves a complex series of steps requiring the expression of genes in both the rhizobial symbiont and the host plant. The necessary genes of rhizobia for nodulation, the nod genes, and for nitrogen fixation, the fix and nif genes, are well studied, nearly all of them having been cloned and characterized. In contrast the symbiosis genes of the legumes are not well understood. A number of plant genes which are specifically expressed in nodules or display enhanced expression in nodules, the so-called nodulin genes, have been identified in pea, soybean, alfalfa and other legumes by mRNA analysis and cDNA cloning. The time of expression of several nodulins has been analyzed in a number of cases and the nodulin genes which are expressed shortly after infection and in the first steps of nodule formation are referred to as early nodulins (ENODs).In addition, a large series of naturally occurring and induced plant mutants, the sym mutants, which have an altered symbiosis, have been described but the sym genes have not been characterized and the functions of the proteins encoded by these genes are not known. Since it seems a reasonable assumption that there will be a limited number of genes involved in nodule formation and metabolism it might well be that some of the sym mutants represent defects in nodulin-coding or controlling sequences. The study presented in this thesis is centred on one of the sym genes, sym2, as we suspected that the sym2 gene has a role in the first interaction between the Rhizobium bacteria and the legume host plant. A typical characteristic of the Rhizobium legume symbiosis is the host specific nature. Most Rhizobium species can nodulate only plants of a specific plant genus. In the recently past years it has been demonstrated that in the Rhizobium bacteria the nod genes are responsible for the strong specificity in the bacteria-plant interaction. The nod genes are involved in the synthesis of specific lipo-oligosaccharides called Nod factors, that can induce the early responses in host plants leading to root nodule formation. Nod factors with a very specific structure are required to induce these responses and they are active at very low concentrations. Therefore it is probable that, as a first step in inducing the reactions leading to nodule formation, the Nod factors are recognized by a special receptor in the host plant. There are now several reasons to presume that the plant sym2 gene might encode the receptor for the specific recognition of Nod factors. The aim of the study presented in this thesis is a further characterization of the interaction between sym2 containing pea lines and different strains of Rhizobium leguminosarium bv. viciae in an attempt to find further support for this hypothesis. At the same time, we have started the detailed genetic mapping of sym2 on the pea genome, in preparation for eventually cloning and molecular characterization of sym2 .In chapter 1 a general introduction summarizes the history of the discovery of the sym2 gene and presents the arguments for the hypothesis that sym2 might code for a receptor of rhizobial Nod factors. In chapter 2 a further genetic characterization of sym2 is given. Chapter 3 describes how a novel early nodulin gene of PsENOD7 was characterized and mapped near the sym2 locus. In chapter 4 a detailed map of the sym2 locus linkage group I is presented including some new molecular markers tightly linked to the sym2 locus. In chapter 5 it is demonstrated how the Rhizobium, nodO gene determines whether sym2 is a dominant or a recessive allele. The thesis ends with some concluding remarks on the nature of sym2 and the impact of the research described in the thesis for the cloning of sym2