Roots systems were fixed with 4% paraformaldehyde and immersed in ClearSee solution for 24 h, as described previously (52 (link)). Forty L. japonicus accessions were screened qualitatively for the formation of nodules, bumps, tumors, and presence/absence of nodule infection 4 wk after R. leguminosarum Norway-GFP inoculation. Ten accessions, which displayed round-shaped nodules, were quantitatively screened for the percentage of infected nodules. Three accessions with either the highest or the lowest number of infected nodules were selected to quantify the area colonized. For each individual accession, 10 nodules were embedded in 6% low melting agarose and sectioned with a VT1000S vibratome (Leica Biosystems) longitudinally. The 50-µm thin sections were visualized with a DM6 B upright microscope (Leica Microsystems). Nodule colonization was calculated for each section as the percentage of infected tissue (nodule cells with GFP signal) relative to the total inner tissue (inwards from the nodule inner cortex). Areas were marked manually in Fiji (53 (link)) and quantified using the default function Measure. Each data point represents the average of the values of three sections from the middle of a nodule.