The non-specific lipid transfer protein N5 of Medicago truncatula is implicated in epidermal stages of rhizobium-host interaction

We show here that MtN5 is a NF-responsive gene expressed at a very early phase of symbiosis in epidermal cells and root hairs. MtN5 expression is induced in vitro by rhizobial effector molecules and by auxin and cytokinin, phytohormones involved in nodule organogenesis. Furthermore, lipid signaling is implicated in the response of MtN5 to rhizobia, since the activity of phospholipase D is required for MtN5 induction in S. meliloti-inoculated roots. MtN5-silenced roots inoculated with rhizobia display an increased root hair curling and a reduced number of invaded primordia compared to that in wild type roots, but with no impairment to nodule primordia formation. This phenotype is associated with the stimulation of ENOD11 expression, an early marker of infection, and with the down-regulation of Flotillin 4 (FLOT4), a protein involved in rhizobial entry.These data indicate that MtN5 acts downstream of NF perception and upstream of FLOT4 in regulating pre-infection events. The positive effect of MtN5 on nodule primordia invasion is linked to the restriction of bacterial spread at the epidermal level. Furthermore, MtN5 seems to be dispensable for nodule primordia formation. These findings provide new information about the complex mechanism that controls the competence of root epidermal cells for rhizobial invasion.Plants belonging to the Leguminosae family have the ability to interact with rhizobia and produce a nitrogen-fixing organ, the root nodule. The symbiotic relationship starts with a molecular cross-talk between the two partners. Host-plant derived molecules are perceived by rhizobia and activate the synthesis of Nod factors (NFs), which, in turn, elicit a variety of biochemical responses in the root hair, including changes in ion fluxes, membrane depolarization, the oscillation of the cytosolic calcium and modification in the cytoskeleton [1-4] that lead to root hair deformation, to infection thread (IT) formation and eventually to the penetration of the bacteri