Targeted inoculation of Medicago truncatula in vitro root cultures reveals MtENOD11 expression during early stages of infection by arbuscular mycorrhizal fungi.

•  An in vitro targeted inoculation technique has been developed for studying the earliest stages of arbuscular endomycorrhizal (AM) infection of Medicago truncatula roots, and in particular the spatio-temporal expression of the early nodulin gene MtENOD11. •  Agrobacterium rhizogenes transformed root explants were derived either from Myc M. truncatula or from the infection-defective Myc mutant TR26 ( dmi2-2 ), both expressing the pMtENOD11-gusA fusion. The normal positive geotropism of these roots, coupled with the negative geotropism of Gigaspora germ tubes allowed oriented growth of the two symbiotic partners, facilitating the identification of initial fungal/root contacts. •  Early infection events at the stage of appressoria and/or internal hyphae could be observed for over 50% of the inoculated explants, revealing that MtENOD11 is expressed transiently in both epidermal and cortical cells at sites of hyphal penetration in Myc roots, but not in epidermal cells in contact with appressoria in Myc roots. •  We propose that a direct link exists between MtENOD11 gene expression and cellular events required for fungal penetration, thereby extending analogies between rhizobial and AM host root infection processes.