Study of Mtn5 transcriptional control and of its involvement in Medicago truncatula nodulation pathway

The symbiosis between legumes and rhizobia starts with an exchange of molecular signals between the two partners. In response to the plant-derived flavonoids, bacteria synthesize Nod Factors (NFs), which are able to induce a series of events, such as ion fluxes, root hair deformation and the expression of the early nodulin genes, that eventually lead to the formation of root nodules. We previously demonstrated that MtN5 is an early nodulin, required for the establishment of the symbiosis and also present in mature nodules. In order to investigate the role of MtN5 in root nodules induction pathway, its expression profile during the early stages of infection was studied. MtN5promoter::GUS fusion showed that the promoter was active in epidermis and root hairs a few hours after inoculation, whilst in mature nodules, GUS was observed in the distal zone.In a time course nodulation experiment, MtN5 showed to be co-expressed with early markers of rhizobia infection, such as RIP1, NIN and ENOD11, and resulted to be more precocious than ENOD20 and MtN6. In transgenic adventitious root silenced for MtN5 expression (MtN5hp roots), we observed that upon rhizobia infection the nodulin MtNIN was not induced, whilst ENOD11 was strongly upregulated with respect to control roots. Furthermore, in MtN5hp roots the expression of FLOT4, a nodulin gene known to be involved in the infection thread growth, was unaffected by the inoculation with symbiotic bacteria, in contrast with what observed in control roots.