Effect of EGTA on the Ca2+ response and nod gene expression induced by L. japonicus exudates. A, M. loti cells were treated with L. japonicus root exudates (black trace) or pretreated with 5 mM EGTA 10 min before adding L. japonicus root exudates (grey trace). B, Top: RT-PCR analysis of control cells (lane 1), cells treated for 1 h with L. japonicus root exudates (lane 2) and cells pretreated with 5 mM EGTA 10 min before treatment with L. japonicus exudates (lane 3). Bottom: Relative percentage of nod gene induction in response to L. japonicus exudates in M. loti cells pretreated (striped bars) or not (black bars) with 5 mM EGTA. Normalization of transcript abundance was done against 16S rRNA. Data are the means ± SEM of three independent experiments. C, Viability, monitored with the BacLight Bacterial Viability kit, of M. loti cells in control conditions or incubated with 5 mM EGTA for 1 h 10 min. As positive control, cells were treated with 70% isopropanol. Live cells fluoresce green, dead cells fluoresce red. Bar = 10 μm. D, Top: RT-PCR analysis of the expression of the housekeeping gene glutamine synthetase II (GSII) in M. loti cells in the absence (-) or presence (+) of 5 mM EGTA. Bottom: Relative transcript abundance of GSII was normalized against 16S rRNA. Bars represent SEM. E, Top: RT-PCR analysis of the inducible aequorin (aeq) gene in M. loti cells in the absence (-) or presence (+) of 5 mM EGTA and 1 mM IPTG. Bottom: Relative transcript abundance of aeq was normalized against 16S rRNA. Bars represent SEM.