Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

EndoPopulus: Elucidation of the Roles of Diazotrophic Endophyte Communities in Promoting Productivity and Resilience of Populus Through Systems Biology Approaches

Authors:

Sharon L. Doty1* (sldoty@uw.edu), Andrew W. Sher1, Robert J. Tournay1, Emma Gomez-Rivas1, Darshi Banan1, Jayde Aufrecht2, Sun Woo Chung1, Matt Hendrickson1, Sriram Parasurama1, Shubroto Sarkar1, Carter Corcoro1, Kevin C. Shaffman1, Amir Ahkami2, Adam Deutschbauer3, Morgan Raimondo1, Clarice Mauer1, P. J. Miller1, Soo-Hyung Kim1

Institutions:

1University of Washington; 2Pacific Northwest National Laboratory; 3Lawrence Berkeley National Laboratory

Goals

The overall project goal is to move toward an understanding of the holobiont, how plants and the microbial community within them interact in ways that promote the productivity of the whole. Integration of plant physiology data with the molecular plant-microbe interactions (multi-omics) data from greenhouse and field experiments will allow researchers to develop a systems-level understanding of the genetic and molecular basis for diazotrophic endophytic mutualism in Populus. This deeper level of understanding of the plant responses will guide construction of microbial communities in order to optimize the impacts of bioinoculants for environmental sustainability of bioenergy crops.

Abstract

The first objective of this project is to unravel the molecular mechanisms of nitrogen fixation by endophytes of Populus. In year 4, researchers published on the intricacies of nitrogenase activity in the aerobic endophyte, Burkholderia strain WPB (Sher et al 2024). Nitrogenase promoter fusions to GFP as well as NanoSIMS with the 15N2-exposed strain indicated that only a subset of the population actively fixes nitrogen (N). Prominent early signatures of N-fixation activity included polyamines and amino acids, while non-N-fixing cells of the population produced more trehalose and citric acid. Poplar plants within RhizoChips were used to assess nitrogenase gene expression in association with the host plant. WPB nitrogenase activity was particularly strong within nitrosomes within the plant root epidermal cells. The wild plant microbiome includes a wide variety of diazotrophic bacterial species, so researchers expanded the research to include Rahnella aceris strain WP5, Azospirillum sp. 11RA, as well as Azorhizobium strain HT1-9, an endophyte of a Hawaiian lava bed plant. The acetylene reduction assay that assesses nitrogenase activity demonstrated that oxygen is required for full activity of 11RA and HT1-9 but that WP5 needed a microaerobic environment. To further elucidate the molecular mechanisms of endophytic nitrogen fixation, researchers have conducted genome-wide transposon-insertion (RB-TnSeq) mutant screening assays to determine genes required for nitrogen fixation at various oxygen levels in WP5 and 11RA. Following up on the initial findings in Burkholderia sp. WPB, researchers have constructed additional fluorescently tagged mutants of both WPB and 11RA to further characterize the nitrosome structures. Researchers continued a detailed analysis of the synergy effect researchers discovered in 2020, uncovering the requirements for the complex microbial interactions that amplify nitrogenase activity.

Greenhouse and field scale experiments were conducted to identify the molecular and physiological impacts on the plant under nutrient-limited conditions (Objective 2) and under water-limited conditions (Objective 3). The physiological impacts are presented in the accompanying poster by Sun Woo Chung. Metabolomics analysis revealed an endophyte-mediated metabolic shift in the leaf tissues. Specifically, potential roles of specific organic acids, amino acids and phytohormones and their correlations with physiological parameters were identified. Verification of plant colonization by each endophyte strain will be done using strain-specific primers in droplet digital PCR. Within Objective 5 on the microbial mechanisms responsible for the plant impacts, the team constructed nitrogenase mutants. In addition to the experiments focused on N-limitation, researchers also initiated more studies on endophyte-mediated phosphate solubilization. Media and protocols are being optimized prior to the mechanistic studies.

References

Sher, A. W., et al. 2024. “Dynamic Nitrogen Fixation in an Aerobic Endophyte of Populus,” The ISME Journal, wrad012. DOI:10.1093/ismejo/wrad012.

Funding Information

This research was supported by the DOE Office of Science, BER Program, grant no. DE-SC0021137.