During arbuscular mycorrhizal (AM) colonization, a focal accumulation of organelles occurs in root epidermal cells, prior to fungal penetration, beneath adhering hyphopodia. This is followed by the appearance of the prepenetration apparatus (PPA), a transcellular column of cytoplasm connected to the nucleus and rich in cytoskeleton and secretory endomembranes. This apparatus appears to be responsible for the construction of an apoplastic compartment that confines the fungus within the cell lumen. To identify AM-specific elements within the PPA response, we challenged root cultures of Medicago truncatula, expressing a green fluorescent protein tag for the endoplasmic reticulum, with an AM symbiont, a necrotrophic pathogen, a hemibiotrophic pathogen, a noncompatible endomycorrhizal fungus, or abiotic physical stimuli. Parallel experiments were made on a M. truncatula nonsymbiotic mutant (doesn't make infections, dmi3-1). The results have highlighted a correlation between physical stimulation of the cell surface and nuclear repositioning. Cytoplasmic aggregation was only induced by contact with compatible fungi, whereas PPA appearance was specifically triggered by the AM fungus. The dmi3-1 mutant did not develop cytoplasmic aggregation or PPA and underwent cell death upon physical stimulation. The up-regulation of an expansin-like gene, already identified as an early marker of AM fungal contact, was triggered in wild-type roots by all the fungi tested. Such observations identify responses that are specific to mycorrhizal interactions and extend the role of the DMI3 protein, a calcium/calmodulin-dependent kinase, from symbiotic to pathogenic interactions.