Fig. 3: Strong MtAnn1-GFP fusion fluorescence and low frequency Ca²⁺ spikes are hallmarks of pre-infection priming in the cortex.

a–e Representative images of rhizobia infection sites in roots co-expressing MtAnn1-GFP (green) and NR-GECO1 (red) in M. truncatula A17. a–c Confocal images illustrate an infected root hair (CFP-labelled rhizobia in the IT, arrow, in magenta) and the nucleus (n) in front, guiding the growth of the IT in a cytoplasmic bridge (arrowhead) (a), the base of the infected root hair cell (b, grey dotted line) and the neighbour C1a-c outer cortical cells (c, dotted lines). C1a-C1c are also shown in (d). d, e Representative Ca²⁺ spiking traces, expressed as signal-to-noise ratio (SNR), from outer cortical cells (C1a-c, C2d-f, C1g-l, top panels) adjacent to two independent infected root hair sites. Cortical cells C1a-c, C2d-f are adjacent to the infection site shown in (a, b) (C1a-c are also shown in c), while C1g-l cells are adjacent to another root hair infection site (not shown). Traces of cortical cells that are in direct contact (C1a, C1b, C1g) or not (C1b-c, C2d-f in d and C1h-l in e) with the infected root hair site are shown. Images were obtained from A17 roots 4 dpi (a–d) or 3 dpi (e) with CFP-labelled S. meliloti. Data were obtained from 2 independent experiments after the analysis of Ca²⁺ traces in n = 12 individual cells. Note that only cortical cells co-expressing MtAnn1-GFP (C1a-b in d, C1g in e) show detectable low frequency Ca²⁺ spiking. Images in a-c are maximal z-projections of sub-stacks and images in d-e are maximal projections of whole time series. Abbreviations: first and second outer cortex layers (C1, C2), infection thread (IT). Scale bars: a–e = 20 µm. See also Supplementary Figs. 3, 6 and 8, 9 for Ca²⁺ spiking responses and MtAnn1-GFP dynamics in primed cells in the cortex in sunn and in infection-defective ern1 and dmi3 mutants. Source data, including split channels for merged fluorescence, are provided as a Source Data file.