Fig. 2: Ca²⁺ spiking amplitude drops along root hair IT development.

a, b Representative bright-field and corresponding confocal fluorescence images of a a root hair with entrapped CFP expressing (magenta)-S. meliloti (RHE) and b a root hair with a growing infection thread (IT) in sunn. Nuclei expressing the NR-GECO1 Ca²⁺ sensor appear in red, and the MtAnn1-GFP fluorescence (green) labels the cytoplasmic zone around the IT and the cytoplasmic bridge connecting and surrounding the nucleus. Representative Ca²⁺ spiking traces of RHE (c) and growing IT stages (d) in sunn. The relative concentration of Ca²⁺ ions in the nucleus is reflected by the intensity of NR-GECO1 fluorescence, expressed as signal-to-noise ratio (SNR, cf. ‘Methods’ section). The number of root hairs with nuclear spiking/total number of root hairs are indicated between parentheses. Nuclei are counted as spiking when showing more than 2 peaks in 10 min. Quantification of nuclear Ca²⁺ spiking: spiking frequency (e), expressed as number of spikes in 10 minutes per nucleus, and spiking amplitude (f), expressed as average SNR of spikes per nucleus. Box plots represent the distribution of individual values (indicated by open circles) from root hairs with entrapped rhizobia RHE, (n = 24 in e and n = 23 in f) or with an IT (n = 21 in e and n = 18 in f) in sunn, 2–4 dpi with S. meliloti from 3 independent experiments. First and third quartile (horizontal box edges), minimum and maximum (outer whiskers), median (centreline), mean (solid black circle) and outliers (crosses) are indicated. Differences were not significant in spiking frequency in IT vs. RHE in (e) (p = 0.1531, two-tailed Mann-Whitney test). Asterisks indicate statistically significant differences in spiking amplitude in RHE vs. IT in (f) (p = 0.0037, two-tailed t-test). Scale bars: a, b = 10 µm. See also Supplementary Fig. 2 and Movies 1, 2 for Ca²⁺ spiking responses in A17 and/or sunn root hairs, and Supplementary Figs. 4–7 for MtAnn1-GFP localisation and fluorescence quantification in root hair cytoplasmic bridges. Source data, including split channels for merged fluorescence, are provided as a Source Data file.