Differential metaproteomics of bacteria grown in vitro and in planta reveals functions used during growth on maize roots

Microbes are ubiquitous in the rhizosphere and play crucial roles in plant health, yet the metabolisms and physiologies of individual species in planta remain poorly understood. In this study, we examined microbial gene expression in response to the maize root environment for seven bacterial species originally isolated from maize roots. We grew each species individually, both in vitro in a minimal medium and in planta, and used differential metaproteomics to identify functions upregulated specifically when bacteria are grown on maize roots. We identified between 1,500 and 2,100 proteins from each species, with approximately 30-70% of these proteins being differentially abundant between the two conditions. While we found that transporter proteins were upregulated in all species in planta, all other differentially abundant functions varied greatly between species, suggesting niche specialization in root-associated microbes. Indeed, in vitro assays confirmed that Curtobacterium pusillum likely degrades plant hemicellulose, Enterobacter ludwigii may benefit the plant by phosphate solubilization, and Herbaspirillum robiniae colonizes maize roots more effectively when both of its Type VI Secretion Systems are functional. Together, our findings highlight both conserved and species-specific bacterial strategies for growth in the root environment and lay a foundation for future work investigating the mechanisms underlying plant-microbiota interactions.