Plant roots establish mutually beneficial interactions with soil microbes, enhancing plant nutrition, pathogen defense, and resilience against adverse environmental conditions. To understand the mechanisms that initiate and stabilize these interactions, we study the molecular dialogue between Arabidopsis thaliana roots and Bacillus, a genus of soil-borne bacteria with several species exhibiting plant growth-promoting and immunomodulatory properties. Utilizing genetically encoded biosensors, we visualize intracellular calcium fluxes and extracellular reactive oxygen species (ROS) formation, providing insights into the spatiotemporal aspects of root cell signaling. Employing microfluidic RootChips, we achieve precise manipulation of the root microenvironment, allowing for the controlled study of signaling dynamics. Structured microdevices that mimic soil architecture enables dissecting the sequential stages of root colonization by B. subtilis, examining the activation and modulation of the plant immune system during this process. This approach has helped us to identify a role of plant defense elements in the structured colonization of roots by beneficial microbes.