EndoPopulus: Elucidating the Molecular Mechanisms of N-Fixation by Populus Endophyte, Burkholderia vietnamiensis WPB
Jayde Aufrecht2*, Andrew Sher1, Daisy Herrera2, Amy Zimmerman2, Young-Mo Kim2, Nathalie Munoz2,Vimal Balasubramanian2, Dusan Velickovic2, Tanya Winkler2, Emma Gomez-Rivas1, Amir H. Ahkami2, and Sharon L. Doty1 (firstname.lastname@example.org)
1University of Washington; and 2Pacific Northwest National Laboratory
The overall goal of this project is to investigate the roles and molecular mechanisms of endophytes in supporting productivity and fitness of Populus. Using systems biology approaches at both lab and field scales, the project will identify the molecular and physiological impacts of the bio-inoculants on the host plant in responding to nutrient and water limitation and determine if bio-inoculants not only increase plant nutrient stores but also “prime” plants for tolerance and resilience to abiotic stresses. Goals include identifying the molecular mechanisms of enhanced plant production and fitness by diazotrophic endophytes. The team will then integrate the plant physiology data with the molecular plant-microbe interactions data to develop a systems-level understanding of the genetic and molecular basis for diazotrophic endophytic mutualism in Populus.
Biological nitrogen fixation (BNF) by microbial diazotrophs can significantly contribute to N availability and uptake in non-nodulating plant species, like Populus spp. There is currently a knowledge gap surrounding the molecular mechanisms underlying plant-diazotroph interactions and the spatial and temporal variations in microbial expression of genes involved in nitrogen fixation. In this work, the team seeks to identify which nitrogenous biomolecules diazotrophs are producing, how BNF is regulated in an axenic culture, and finally how Populus trichocarpa regulates N fixation during co-culture with diazotrophic endophytes. Through a 15N2 time course enrichment study, researchers identified key nitrogenous metabolites and proteins that are synthesized by diazotroph Burkholderia vietnamiensis (WPB). Using a fluorescent transcriptional reporter in the nitrogen fixing gene, nifH, researchers found that nifH is not uniformly expressed across genetically identical colonies of WPB. This result led to conducting a follow-on targeted metabolomics study in colony sections with and without nifH expression to identify which of the key nitrogeneous metabolites are produced in each scenario. Although WPB does not require the host plant to fix N, it was hypothesized that the plant can regulate N fixation via metabolite exchange with the diazotroph under environmental changes including water-limiting conditions. Using liquid chromatography–mass spectrometry, researchers have identified 39 compounds in P. trichocarpa root exudates that are differentially abundant in a drought vs. well-watered condition. Currently, the team is testing the influence of these root exudates on the differential gene expression of WPB (including nifH gene) in axenic culture. Additionally, researchers have cultured WPB with the host plant directly in the RhizoChip, a synthetic soil habitat, which enabled direct imaging of the expression of microbial nifH within root epidermal cells. The team found that nifH expression is heterogeneous within root tissues, depends on the presence of soluble N compounds, and is localized to the root elongation zone where the WPB forms a unique physical interaction with the root cells. Finally, to understand the spatial distribution of metabolites exchanged by the plant, matrix-assisted laser desorption ionization–mass spectrometry imaging (MALDI-MSI) was used to image the distribution of various hormones and metabolites in root cross sections in plants subjected to drought vs. a well-watered condition. This experiment was repeated with plants that were inoculated with a community of endophytes to determine how the presence of endophytes alters the internal molecular environment of the root and how abiotic stressors like drought affect these interactions. These comprehensive experiments merge multiomics, chemical, and optical imaging data from axenic microbial, plant, and co-cultures to identify the key molecular mechanisms regulating beneficial plant-endophyte interactions.
This research was supported by the DOE Office of Science, Biological and Environmental Research (BER) Program, grant no. DE-SC0021137.