g. proteins [30, 31] or plant cell wall fragments released during TPCA-1 nmr the detachment of border cells from the root tip , activating a different Ca2+ signalling pathway. Further confirmation of the specificity of the host plant-induced Ca2+ signalling comes from the complete absence of any detectable Ca2+ change and nod gene
transcriptional activation by root exudates from a non-legume (tomato) (Fig. 4A and 4B). Figure 4 Monitoring [Ca 2+ ] i and nod gene expression in response to non-host legume and non-legume root exudates. Bacteria were challenged with root exudates from soybean (A, black trace; B, lane 2), V. sativa subsp. nigra (A, grey trace; B, lane 2) and tomato (A, light grey trace; B, lane 2). Control cells were treated with cell culture medium only (B, lane 1). Discussion Even though Ca2+-based signal transduction processes are well-established selleck to underpin plant cell responses to rhizobial informational molecules, a possible involvement of Ca2+ as a messenger in rhizobia in response to plant symbiotic signals has not hitherto been considered. We approached this issue by constructing a M. loti strainexpressing the bioluminescent Ca2+ indicator aequorin. The highly sensitive and reliable aequorin-based method is widely used to monitor the dynamic changes of [Ca2+]i in both eukaryotic  and bacterial [18, 16] living cells and represents to date
the tool of choice for monitoring Ca2+ changes in cell populations . The effectiveness of this recombinant technique has been verified at more than one level, and the results obtained demonstrate the utility of aequorin as a probe to study the early recognition events in rhizobium-legume interactions from the bacterial perspective. The generation of a well-defined and reproducible Ca2+ transient in M. loti cells in response to root exudates of the host plant L. japonicus containing nod gene inducers is indicative of
Ca2+ participation in sensing and transducing Casein kinase 1 diffusible host-specific signals. It cannot be ruled out that the biphasic this website pattern of the Ca2+ trace (Fig. 2B), monitored by the aequorin method, may be due to an instantaneous synchronized Ca2+ increase in cells immediately after stimulation, followed by a sustained Ca2+ response probably due to the sum of asynchronous oscillations occurring in single cells. Ca2+ oscillations, considered as a universal mode of signalling in eukaryotic cells [34–36] have been proposed to occur in bacteria as well . The significant inhibition of nod gene expression obtained when the Ca2+ elevation is blocked indicates that an upstream Ca2+ signal is required for nod gene activation. The Ca2+ dependence of nod gene expression strongly suggests that the [Ca2+]i change, evoked by L. japonicus exudates, represents an essential prerequisite to convey the plant symbiotic message into rhizobia.