PNNL

Abstract

Biological nitrogen fixation (BNF) by microbial diazotrophs can contribute significantly to nitrogen availability and uptake in non-nodulating plant species. However, there is a knowledge gap currently surrounding the molecular mechanisms underlying diazotrophic endophyte-plant interactions and the spatial and temporal variations in microbial gene expression relating to BNF.The aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, was previously isolated from wild Populus trichocarpa and in this study served as a model for diazotrophic endophyte-poplar interactions. Growth on nitrogen-free medium indicated that nitrogen-fixing activity was dynamic, with some cells growing to form robust, raised globular like colonies while others did not. Incubation with 15 N2 gas followed by nanoscale secondary ion mass spectrometry (NanoSIMS) confirmed that nitrogen fixation is uneven within the population.Using a fluorescent transcriptional reporter (GFP) in the nitrogenase subunit gene, nifH, we found that the nifH gene is not uniformly expressed across genetically-identical colonies of WPB. Using fluorescence-activated cell sorting (FACS), it was estimated that only approximately 11% of the population was actively expressing the nitrogenase gene. Higher gene expression was observed in clustered cells through the monitoring of individual bacterial cells through single molecule fluorescence in-situ hybridization (smFISH) targeting mRNA of nifH. Through a 15 N2 time course enrichment study, we identified key nitrogenous metabolites and proteins that are synthesized by WPB under N-limited conditions, and then employed targeted metabolomics to assess nifH active and nifH inactive sectors. We co-cultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of the expression of microbial nifH within root epidermal cells. The nitrogenase gene was also expressed while in association with the poplar plant, demonstrating that the required conditions for nitrogen fixation were met. We observed that nifH expression is heterogeneous within root tissues and is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. In this work, we employed comprehensive experimentation merging multi-omics, chemical, and optical imaging data from axenic microbial, plant, and co41 cultures to identify key and novel molecular mechanisms regulating beneficial plant-endophyte interactions.