a Representative micrograph of roots expressing the indicated receptor variants. YFP: yellow fluorescent protein (transformation marker), M. loti DsRed: fluorescent M. loti R7a, pNin:GUS: Bright Field photos after GUS staining. Scale bars: 5 mm. b Nodule primordia formation in nfr1-1 pNin:GUS roots expressing the indicated receptor variants. We observe that expression of distinct receptor versions induced many organogenetic events inside the roots (red asterisks in a) which did not develop at the time of analysis (5 weeks post-inoculation) into nodules (black asterisks in a) that protrude out of the roots. Nonparametric Kruskal–Wallis test was performed to assess differences among receptor variants for the formation of nodule primordia (p = 1.46 × 10−12). Unique lower-case letters above whiskers note significant differences (Dunn’s test, adjusted with Benjamini–Hochberg method, p < 0.05) among receptor variants. c Sections of nodules formed by nfr1-1 roots expressing the full-length receptor (1) and the receptor variant 28 (white nodule) 5 weeks post-inoculation with M. loti R7a DsRed. Scale bars: 1 mm. d Number of infection chambers (ICs) or infection threads (ITs) formed by nfr1-1 roots expressing the indicated receptor variants. Infection events counted after LacZ staining 2 weeks post-inoculation with M. loti R7a DsRed. Fractions beneath the box plots show plants that formed infection events out of the total plants analysed. Nonparametric Kruskal–Wallis tests were performed to assess differences among receptor variants for each trait (ICs, p = 0.033; ITs, p = 3.10×10−8). Unique lower-case letters above whiskers note significant differences (Dunn’s test, adjusted with Benjamini–Hochberg method, p < 0.05) among receptor variants. e,f Representative micrographs of root hair responses (e) and infection events (f) of nfr1-1 roots expressing the indicated receptor variants (EV-empty vector). Black arrowheads indicate root hair deformation or root hair curling. Scale bars: 30 μm. g Representative micrographs of epidermal NIN activation of nfr1-1 pNin:GUS roots expressing the indicated receptor variants. Note the unspecific GUS staining in the inner root tissue at the points of lateral root emergency. This differs from the NIN-GUS staining in the epidermis. Receptors 27 and 28 show symbiotic capacities when analysed at later time point (5 weeks post-inoculation- see Fig. 4b). Fractions on the top left corner indicate the number of plants in which NIN activation was observed out of the total number of analysed plants. Scale bars: 2 mm. h Number of pink/fixing or white/non-fixing nodules41 formed by roots expressing the indicated receptor variants. Nodules were counted 5 weeks post-inoculation with M. loti R7a DsRed. Fractions beneath the box plots show nodulating plants out of the total plants analysed. Nonparametric Kruskal–Wallis tests were performed to assess differences among receptor variants for each trait (pink nodules, p < 2.2 × 10−16; white nodules, p < 2.2 × 10−16). Unique lower-case letters above whiskers note significant differences (Dunn’s test, adjusted with Benjamini–Hochberg method, p < 0.05) among receptor variants.
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