Perception and action of nod factors in Rhizobium-legume symbiosis.

Rhizobium bacteria are able to invade the roots of their leguminous hosts and trigger the formation of a new organ, the root nodule. In these nodules the bacteria are hosted in the proper environment for fixing atmospheric nitrogen into ammonia, making plant growth independent of nitrogen compounds from the soil. The interaction between the bacterium and the host plant starts with a signal exchange, when flavonoids excreted by the plant induce the transcription of bacterial nodulation (nod) genes. Induction of these nod genes leads to the synthesis of specific lipo-oligosaccharides (Nod factors) required for inducing various root responses like root hair deformation, infection thread and nodule primordia formation. The aim of the research described in this thesis is to contribute to the insight in the mechanism by which Nod factors initiate root nodule formation. To address this issue it is essential to have a plant system in which both the morphological and molecular changes induced by Nod factors can be studied. We adapted a root hair deformation assay for Vicia sativa (vetch), growing the seedlings in Fahraeus slides, to study the activity and fate of the various Nod factors secreted by Rhizobium leguminosarum by viciae in a fast, simple and semiquantitative way (Chapter 2). Root hair deformation in this assay is limited to root hairs in a specific developmental stage and involves a reinitiation of tip growth which is visible within 3 hours after Nod factor application. In an attempt to identify genes the expression of which is induced in an immediate respons to Nod factors we used the differential RNA display method to compare the pattern of cDNAs expressed in the zone of the vetch root where root hair deformation occurs at 0, 1 and 3 hours after Nod factor addition. Surprisingly, the sequence of one of the isolated clones already expressed I hour after Nod factor application coded for leghemoglobin. This gene was used to show that gene expression and root hair deformation are not coupled and to investigate how NH4NO3* might block root hair deformation (Chapter 3). Root hair deformation induced by Nod factors involves a reinitiation of growth in the existing root hair tip, a mechanism reminiscent of ethylene induced tip growth leading to the formation of root hairs in the epidermis. Since ethylene is a potent inhibitor of cortical cell division there seems to be a paradox in its action during the initial interaction between rhizobia and its host. We showed that ethylene is not invoved in the root hair deformation process, but it is one of the factors involved in giving positional information determining where nodule primordia can be induced (Chapter 4). How Nod factors are perceived and induce the various responses in the root is poorly understood. Considering that Nod factors are active at very low concentrations it is likely that they are recognized by receptors. Besides a biochemical approach to search for such receptors it win be very important to isolate and analyse host mutants disturbed in the early staves of the interaction with Rhizobium. We chow to focus on the sym2 ^A gene originating from Afghanistan pea since the presence of this gene puts more stringent structural demands on the Nod facotrs secreted by Rhizobium leguminosarum by viciae. After detailed analysis of the phenotype conferred by sym2 ^A we propose that Sym2 controls the infection process in the epidermis, possibly representing a Nod factor receptor (Chapter 5)