Development of an Experimental Approach to Achieve Spatially Resolved Plant Root-Associated Metaproteomics Using an Agar-Plate System

Plant-microbe interactions in the rhizosphere play a vital role in plant health and productivity. The composition and func-tion of root-associated microbiomes is strongly influenced by their surrounding environment, which is often customized by their host. How microbiomes change with respect to space and time across plant roots remains poorly understood, and method-ologies that facilitate spatiotemporal metaproteomic studies of root-associated microbiomes are yet to be realized. Here, we developed a method that provides spatially resolved metapro-teome measurements along plant roots embedded in agar-plate culture systems, which have long been used to study plants. Spa-tially defined agar plugs of interest were excised and sub-sequently processed using a novel peptide extraction method prior to metaproteomics, which was used to infer both microbial community composition and function. As a proof-of-principle, a previously studied 10-member community constructed from a Populus root system was grown in an agar plate with a 3 -week-old Populus trichocarpa plant. Metaproteomics was per-formed across two time points (24 and 48 h) for three distinct locations (root base, root tip, and a region distant from the root). The spatial resolution of these measurements provides evidence that microbiome composition and expression changes across the plant root interface. Interrogation of the individual microbial proteomes revealed functional profiles related to their behavioral associations with the plant root, in which chemotaxis and augmented metabolism likely supported predominance of the most abundant member. This study demonstrated a novel peptide extraction method for studying plant agar-plate culture systems, which was previously unsuitable for (meta)proteomic measurements.

Automated summary

Plant-microbe interactions in the rhizosphere are important for plant health and productivity. This study developed a method for spatially resolved metaproteomic analysis of root-associated microbiomes in agar-plate culture systems, providing insights into microbial community composition and function.