Abstract
Overproduction of indole-3-acetic acid (IAA) by rhizosphere bacteria disrupts auxin homeostasis and induces root growth inhibition (RGI) in plants. Bacteria from the genus Variovorax mitigate this effect by degrading IAA, and in our prior work we identified the iad locus as being required for this activity. Here, we refine our understanding of the iad pathway using bacterial genetics, metabolomics, and isotope tracing to assign roles to individual Iad pathway enzymes and show that IadDE, though resembling a Rieske dioxygenase, functions instead as a monooxygenase that initiates catabolism via a novel intermediate. Guided by these insights, we installed chromosomal iad cassettes into root-associated commensals (Polaromonas MF047 and Paraburkholderia MF376), creating the first engineered bacteria that reprogram rhizosphere auxin homeostasis in microbially complex environments to benefit the plant. In natural soil, engineered Paraburkholderia enhanced plant biomass, and community profiling revealed no significant differences in microbiome composition between engineered and wild type treatments, supporting that auxin degradation conferred plant benefit without broader disruption of the rhizosphere community. Together, this work refines the pathway logic of microbial auxin degradation and demonstrates that commensals can be rationally engineered to deliver auxin-balancing functions in complex rhizosphere microbiomes. More broadly, it provides a framework for leveraging mechanistic insight to engineer plant-associated commensals that enhance plant growth, laying the foundation for deployment in agricultural settings.