Garrell, A.-K. | Search bioRxiv

bioRxiv2025

A Minimal Medium for Culturing Maize Root-Associated Microbes Based on a Plant Growth Medium

Plant-associated microbiota play a critical role in host resilience to both abiotic and biotic stresses. However, understanding the underlying mechanisms of interaction within these communities, as well as between these communities and their hosts, remains challenging due to the complexity and dynamic nature of plant-associated microbiota. Synthetic microbial communities (SynComs) can serve as experimentally tractable models to establish a fundamental understanding of plant-microbiota interactions. Here, we report the development of a defined, minimal growth medium for a well-characterized seven-member maize root SynCom. The medium is based on a standard plant-growth medium to enable its use for both in vitro and in planta studies. Using genome-scale metabolic modeling and auxotrophy prediction, we identified key nutrient requirements of each of the seven species and optimized the medium composition to support bacterial growth. This minimal medium enables controlled investigation of microbial physiology, metabolite exchange, and community interactions, and lays the foundation for scalable in vitro and in planta experiments, facilitating future research on plant microbiome functions.

microbiology↗

bioRxiv2025

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 proteomics 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 20-60% 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. ImportanceBacteria that live on and around plant roots are important for plant growth and health, yet we still know relatively little about how individual bacterial species behave in this environment. In this study, we looked at seven bacterial species originally isolated from maize roots to understand how they change their metabolism and physiology when grown on the plant versus when grown under laboratory conditions. By doing this, we identified key strategies bacteria use to survive and thrive in the root environment, including changes in nutrient uptake, metabolism, and secretion systems. We also substantiated some of these behaviors using lab experiments and bacterial mutants. Understanding these species-specific functions helps us learn how bacteria establish themselves on roots and interact with the plant. This knowledge is critical for future efforts to design effective microbial communities that improve crop performance and resilience.

microbiology↗