Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Plant-Microbe Interfaces: Unraveling How Microbial Adaptation Strategies Are Shaped by the Chemical Environment of the Populus Rhizosphere


Robert Hettich* (, Sameer Mudbhari, Manasa Appidi, Dana Carper, Amber Bible, Jenny Morrell-Falvey, Tomas Rush, Paul Abraham, Mitchel Doktycz


Biosciences Division, Oak Ridge National Laboratory



The overriding goal of the Oak Ridge National Laboratory (ORNL) Plant-Microbe Interfaces (PMI) Science Focus Area is to predictively understand the productive relationship between a plant host and its microbiome based on molecular and environmentally defined information. Populus and its associated microbial community serve as the experimental system for understanding this dynamic, complex multi-organism system. To achieve this goal, researchers focus on: (1) defining the bidirectional progression of molecular and cellular events involved in selecting and maintaining specific, mutualistic Populus-microbe interfaces; (2) defining the chemical environment and molecular signals that influence community structure and function; and (3) understanding the dynamic relationship and extrinsic stressors that shape microbiome composition and affect host performance.


The rhizosphere microbiome for Populus is critically important but factors driving its formation, maintenance, and stability are poorly understood. This inter-kingdom interaction is connected and controlled by molecular information, primarily metabolites and proteins, exchanged between the two systems. As such, these biomolecules are key indicators of the dynamic and spatial functioning of these interactions. Based on this need, researchers have optimized liquid chromatography-tandem mass spectrometry approaches for deep, agnostic measurement of microbial proteomes and metabolomes in the rhizosphere in an effort to explore how bacteria adjust their metabolic activities in response to the chemical environment of the Populus rhizosphere.

In order to explore the fundamental interactions of the plant-microbiome, it is advantageous to employ a lab-based system that has lower complexity than field-based systems. To this end, researchers have examined the interaction of a 10-member customized bacterial community interacting with plant roots, all maintained in an agarose gel plate (Appidi et al 2022). This enables the detection of microbial proteomes and metabolomes in a temporal and spatial fashion as the microbes encounter and interact with the plant. By employing a metaproteomic approach, researchers contrasted how each of these microbes responds to the plant when grown individually versus how the microbial consortium responds to the plant as a community. Researchers found that while the microbial response is highly individualized, there are some common responses between the isolates and the microbial community that were shared and included the upregulation of proteins associated with chemotaxis, motility, transporters, and metabolism. Metabolomic measurements revealed a large array of primary and secondary metabolites, many of which vary temporally in the plant-microbiome system. Workflows such as Compound Discoverer are being used for putative molecular identifications, although many detected compounds elude annotation. One approach to deal with the molecular complexity is to employ molecular networking approaches such as GNPS to connect similar fragmentation spectra. This enables experimental MS/MS spectra to be matched against a large collection of publicly accessible natural product and metabolomics fragmentation data in the GNPS-community spectral library to assign putative annotations and identify molecular families, which are defined as related MS/MS spectra differing by simple structural or chemical transformations.

The key information obtained above will provide the springboard to extend to the more complex field-systems. To address the expected challenges, researchers have focused attention on key advances in sample preparation techniques (to reduce complexity in the samples), MS measurement features, and bioinformatic data-mining (Carper et al 2022). The most recent work has been to evaluate the newly emerging series of commercially available rapid-scanning mass spectrometers. In particular, researchers evaluated a synthetic microbial community sample on the new ThermoFisher Astral MS platform, which scans at least 20X faster than existing instrumentation. As expected, even with the same sample prep process, the measurement metrics are about an order of magnitude improved, which equates to greater microbial proteome coverage. This experimental platform should dramatically advance the utility of examining more complex in situ microbiomes from field-sampling campaigns and thus provide more detailed information about plant-microbial interactions at the environmental level.


Appidi, A. R., et al. 2022. “Development of an Experimental Approach to Achieve Spatially Resolved Plant Root-Associated Metaproteomics Using an Agar-Plate System,” Molecular Plant Microbe Interactions 35(8) 639–49. DOI:10.1094/MPMI-01-22-0011-TA.

Carper, D. L., et al. 2022. “The Promises, Challenges, and Opportunities of Omics for Studying the Plant Holobiont,” Microorganisms 10(10), 2013. DOI:10.3390/microorganisms10102013.

Funding Information

Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the U.S. DOE under contract no. DE-AC05-00OR22725. The Plant-Microbe Interfaces Science Focus Area is supported by the U.S. DOE, Office of Science, through the GSP, BER Program under FWP ERKP730.