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Plant roots use exudates to assemble microbiomes that in turn influence the function and stress resilience of plants. Yet, how specific exudates drive spatial colonization patterns remains largely unknown. Here, we demonstrate that endodermal Casparian strips, forming the root extracellular diffusion barrier, restrict nutrient leakage into the rhizosphere, coinciding with and controlling spatial colonization patterns of rhizobacteria. We find that vasculature-derived glutamine is a major bacterial chemoattractant and enhancer of proliferation, defining a novel leakage pathway for root exudate formation. Bacteria defective in amino acid chemoperception display reduced attraction towards leakage sites, and roots with Casparian strip defects display bacterial over-proliferation, dependent on bacterial capacity for amino acid metabolization. An associated, chronic immune stimulation suggests that endodermal nutrient restriction is crucial for beneficial microbial colonization.