Spatiotemporal changes in cellular calcium (Ca2+) concentrations are essential for signal transduction in a wide range of plant cellular processes. In legumes, nuclear and perinuclear-localized Ca2+oscillations have emerged as key signatures preceding downstream symbiotic signaling responses. Förster resonance energy transfer (FRET) yellow-based Ca2+cameleon probes have been successfully exploited to measure the spatiotemporal dynamics of symbiotic Ca2+signaling in legumes. Although providing cellular resolution, these sensors were restricted to measuring Ca2+changes in single subcellular compartments. In this study, we have explored the potential of single fluorescent protein-based Ca2+sensors, the GECOs, for multicolor and simultaneous imaging of the spatiotemporal dynamics of cytoplasmic and nuclear Ca2+signaling in root cells. Single and dual fluorescence nuclear and cytoplasmic-localized GECOs expressed in transgenicMedicago truncatularoots andArabidopsis thalianawere used to successfully monitor Ca2+responses to microbial biotic and abiotic elicitors. InM. truncatula, we demonstrate that GECOs detect symbiosis-related Ca2+spiking variations with higher sensitivity than the yellow FRET-based sensors previously used. Additionally, in bothM. truncatulaandA. thaliana, the dual sensor is now able to resolve in a single root cell the coordinated spatiotemporal dynamics of nuclear and cytoplasmic Ca2+signalingin vivo. The GECO-based sensors presented here therefore represent powerful tools to monitor Ca2+signaling dynamics invivoin response to different stimuli in multi-subcellular compartments of plant cells.