Early nodulin gene expression and the action of nod factors in Vicia sativa

Bacteria of the genera Rhizobium, Bradyrhizobium or Azorhizobium secrete lipo-oligosaccharide signal molecules, which play a pivotal role in the induction of early steps of root nodule formation on legumes. In these nodules the bacteria are hosted and a proper environment is provided for the bacteria to fix atmospheric nitrogen into ammonia, making the plant for its growth independent of nitrogen compounds in the soil.The goal of the research described in this thesis is to provide insight in the mechanism by which the Nod factors secreted by Rhizobium bacteria initiate root nodule formation. Such kind of study requires a plant-bacteria system in which both the morphological and molecular changes induced by the Nod factor can be examined. Working on the Nod factors secreted by Rhizobium leguminosarum bv. viciae the choice of the host plant is limited to the genera Pisum, Lathyrus, Lens and Vicia. We decided that Viciasativa (vetch) would be the most useful plant for our studies, since the plant is small and various morphological changes, like root hair deformation and the formation of nodule primordia, that are induced by the Nod factors can easily be observed (Chapter 1).Nod factors can induce the expression of early nodulin genes. These genes are expressed during different developmental stages of root nodule formation and the expression of these genes can therefore be used as molecular markers of root nodule development and Nod factor induced processes. To study Nod factor induced plant responses in V.sativa on the molecular level, the homologues of the early nodulin genes of pea (Pisum sativum) were isolated from V. sativa and their expression pattern was studied by in situ hybridization during root nodule development (Chapter 2).To obtain information about the pathway from Nod factor to early nodulin gene expression, the mechanisms controlling ENOD12 expression were studied. With a promoter analysis in transgenic V.hirsuta root nodules we have identified that the 200 bp immediately upstream of the transcription start are sufficient to induce nodule specific and Nod factor induced expression (Chapter 3). For the isolation of transcription factors involved in controlling ENOD12 expression an expression library was screened and a preliminary characterization of cDNA clones encoding polypeptides that bind to the PsENOD12 promoter is described in Chapter 4.In Chapter 5 early nodulin gene expression during Nod factor induced morphological changes, like root hair deformation and nodule primordia induction, was studied. Furthermore we examined whether RNA and protein synthesis are required for root hair deformation and for the activation of the early nodulin genes. These studies provided new insights about the mode of action of Nod factors.In Chapter 6 is discussed to what extent V. sativa is a suitable host plant to study the mode of action of Nod factors and in which way the studies reported in this thesis have contributed to elucidate the mechanism by which Nod factors induce a diversity of plant responses.