Native poplar (Populus) plants have a diverse microbiota including endophytes, microorganisms that live within plants, some of which can fix atmospheric nitrogen (N2) into forms of nitrogen (N) available to their host plant. These beneficial endophytes promote plant growth and health under abiotic stresses. Our lab has demonstrated using the 15N2 incorporation assay that N2 is fixed at high levels in wild poplar by endophytes, with varying levels in different cuttings. Addition of a group consisting of 6 strains of endophytes to young hybrid poplar resulted in increased growth and N-fixation. To successfully apply endophyte technologies to increase overall biomass produced by bioenergy plantations, it is necessary to have a greater understanding of the mechanisms by which endophytes provide fixed N to their host. The proposed project will use the emerging 'omics capabilities of EMSL in synthetic communities in vitro to determine which genes are co-expressed specifically under conditions conducive to N-fixation. Following identification of the key genes required, we propose to use EMSL's fluorescence in situ hybridization capabilities to determine the spatiotemporal bacterial colonization and N-fixation activity patterns within endophyte-inoculated poplar. In parallel, with the ultimate goal of identifying the key players involved in N-fixation from wild Populus, we will use laser capture microdissection microscopy and metatranscriptomics. Identification of the most expressed N cycle genes will provide the needed guidance to select the most active diazotrophic bacteria from wild poplar microbiota for use in sustainable bioenergy plantations.