Nodulin gene expression in the developing pea root nodule

Infection of leguminous plants with bacteria of the genus Rhizobium results in a symbiotic interaction which brings about the development of an entirely new organ on the plant, the root nodule. Within this organ about half of the plant cells are inhabited by bacteroids, the endosymblotic form of the bacterial partner. The bacteroids reduce atmospheric nitrogen (N ₂ ) to ammonia (NH ₃ ) which the plant uses as a nutrient. The host plant, on the other hand, provides the rhizobia with carbon compounds as an energy source for the nitrogen fixation process. Development of a nitrogen fixing root nodule is a true co-operation between two genomes. In chapter II the plant and bacterial genes involved in this symbiotic interaction are reviewed.

The research reported in this thesis deals with the identification of plant genes which are specifically expressed during the development of the symbiosis, the so-called nodulin genes and, in addition, bears upon the question which bacterial genes are involved in regulating the expression of nodulin genes.

By comparing the RNA content of pea root nodules induced by Rhizobiumleguminosarum and uninfected pea roots by means of invitro translation, we have been able to demonstrate the expression of twenty one nodulin genes (chapter III). Out of a nodule cDNA library seven pea nodulin cDNA clones were selected (chapter IV). The appearance of nodulin mRNAs during pea root nodule development, studied by analyses of invitro translation products and Northern blots, showed that nodulin genes are differentially expressed. Thus, nodulins could be classified into two groups: class I or early nodulins and class II nodulins which are expressed later in nodule development (chapter III and IV).

To determine the origin and the nature of signals responsible for the induction of nodulin genes, we mimicked the microaerobic conditions supposed to exist in root nodules and showed that in pea roots microaerobiosis by itself is not sufficient to trigger the induction of nodulin gene expression (chapter V). Also, in pea tumors formed by Agrobacteriumtumefaciens no expression of nodulin genes was detected (chapter VI). Therefore, it seems likely that nodulin gene expression is induced by specific signals from Rhizobium and we have searched for the Rhizobium genes responsible for such signals.

Analysis of nodulin gene expression in non-effective nodules, formed by Rhizobium mutants and engineered RhizobiumandAgrobacterium strains, showed that Rhizobium genes required for nodulation ( nod genes) are involved in the induction of at least one early nodulin gene (chapter VI), whereas Rhizobium genes essential for the nitrogen fixation process ( nif and fix genes) are not required for the induction of nodulin gene expression (chapter III IV and VI). In noneffective nodules the final accumulation level of class II nodulin mRNAs is reduced, indicating that the gene products of the Rhizobiumnif and fix genes, directly or indirectly via the products of the nitrogen fixation process, regulate the level of nodulin gene expression.

Finally, in chapter VII the implications of the reported results are discussed in the perspective of the possible functions of nodulins in the developing root nodule and the way nodulin gene expression may be induced and regulated.