Development of an experimental approach to achieve spatially resolved plan root-associated metaproteomics using an agar-plate system
Manasa R. Appidi, Amber N. Bible, Dana L. Carper, Sara S. Jawdy, Richard J. Gianonne, Robert L. Hettich, Jennifer L. Morrell-Falvey and Paul E. Abraham
22-March-2022, Molecular Plant-Microbe Interactions; https://doi.org/10.1094/MPMI-01-22-0011-TA
Plant-microbe interactions in the rhizosphere play a vital role in plant health and productivity. The composition and function 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 methodologies that facilitate spatio-temporal metaproteomic studies of root-associated microbiomes are yet to be realized. Here, we developed a method that provides spatially resolved metaproteome measurements along plant roots embedded in agar-plate culture systems, which have long been used to study plants. Spatially defined agar ‘plugs’ of interest were excised and subsequently 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 P. trichocarpa plant. Metaproteomics was performed across two time points (24-h, 48-h) for three distinct locations (root base, root tip, and a region distant from the root). The spatial resolution of these measurements provide 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.