The <i>gac</i> system integrates physical and chemical cues to promote plant root attachment

ABSTRACT Plants host complex communities of microbes that are attached to root surfaces. While many studies have sampled mutant populations after prolonged incubation on roots to identify bacterial genes that enable long-term colonization, the molecular mechanisms governing the early stages of root attachment remain less understood. Here, we developed an in vitro root culture system that enables controlled and scalable investigation of bacterial attachment to root tissue. We used this platform to perform a genome-wide screen for root attachment determinants in the plant-associated bacterium Pseudomonas protegens Pf-5. Our results reveal that the gacSA two-component system functions as a sensory integration hub for coordinating early root attachment. Mutations that disrupt gacS or gacA cause severe root attachment defects despite having no effect on abiotic surface attachment in standard biofilm assays. Mutation of flagellar assembly genes enhances root attachment by mimicking surface contact and activating the gac system. In parallel, chemical cues released by roots stimulate surface attachment in a gac- dependent manner. By integrating these signals, the gac system activates cyclic-di-GMP-mediated attachment programs that drive the transition from planktonic to sessile behavior required for root association. We build on this model to show that manipulating flagellar surface-sensing sustains the attachment of Pf-5 in the presence of a synthetic bacterial community, suggesting a strategy to improve the attachment of beneficial microbes on crops. These findings establish a mechanistic framework linking surface sensing, global regulation, and root attachment in a beneficial rhizobacterium. IMPORTANCE Plant roots are covered with diverse microbes that strongly influence plant health. Growing in association with roots has many benefits, but how bacteria attach to root tissue remains poorly understood. We developed a system to study how the plant-associated bacterium Pseudomonas protegens Pf-5 attaches to roots. We found that physical contact with the root surface and chemical cues released by roots both enhance attachment to roots. A sensory system called gacSA is responsible for integrating physical and chemical cues to activate a root attachment program. Variant bacteria that prematurely activate the gac system attach more effectively even in the presence of other bacteria on roots, suggesting that the root attachment pathway we characterized could serve as a strategy to use beneficial bacteria in agriculture.