Signal transduction enables plants to detect and respond to environmental stress. Calcium (Ca2+) and Reactive Oxygen Species (ROS) are key signaling molecules in plants, regulating plant immunity and programmed cell death. However, the specificity of Ca2+ and ROS signaling responses remains unclear. We established a bidirectional dual-flow RootChip microfluidic device to expose G-CaMP3 and HyPerGFP A. thaliana primary roots to asymmetric solute gradients. We utilized linear one-sided concentration gradients for PEG, NaCl, and Pathogen-Associated Molecular Patterns Flg22 and PEP13. Additionally, we infected A. thaliana roots with root rot inducing pathogen Phytophthora cinnamomi spores and mycelium. Fluorescent live-cell imaging was used to detect and quantify cytosolic Ca2+ and ROS in parallel to root growth measurements, and spore localization. Importantly, the plants' Ca2+ and ROS spatiotemporal signal patterns in response to treatments are significantly different and indicate cell-type-specific response mechanisms. To gain further insight into the impact of osmotic stress on the host-pathogen interaction both, the plant and pathogen, were treated with NaCl and PEG followed by a label-free quantitative proteomics approach. Results indicate that P. cinnamomi has a host-comparable stress adaptation strategy, such as upregulation of ROS quenching. Furthermore, pathogenicity proteins, such as RxLR and CRN effector proteins, are significantly increased 12 hours after stress treatment. A computational approach was chosen for network analysis, 3D structural modeling, and protein conservation studies. We will further discuss specific proteins and their potential role in plant infection and increased pathogenicity during drought and osmotic stress conditions.