In situ immunolocalization of tubulin revealed that important rearrangements occur during all the early symbiotic steps in the Medicago/R. meliloti symbiotic interaction. Microtubular cytoskeleton (MtC) reorganizations were observed in inner tissues, first in the pericycle and then in the inner cortex where the nodule primordium forms. Subsequently, major MtC changes occurred in outer tissues, associated with root hair activation and curling, the formation of preinfection threads (PITs) and the initiation and the growth of an infection network. From the observed sequence of MtC changes, we propose a model which aims to better define, at the histological level, the timing of the early symbiotic stages. This model suggests the existence of two opposite gradients of cell differentiation controlling respectively the formation of division centers in the inner cortex and plant preparation for infection. It implies that (i) MtC rearrangements occur in pericycle and inner cortex earlier than in the root hair, (ii) the infection process proceeds prior to the formation of the nodule meristem, (iii) the initial primordium prefigures the future zone II of the mature nodule and (iv) the nodule meristem derives from the nodule primordium. Finally, our data also strongly suggest that in alfalfa PIT differentiation, a stage essential for successful infection, requires complementary signaling additional to Nod factors.

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