EndoPopulus: Elucidation of the Roles of Diazotrophic Endophyte Communities in Promoting Productivity and Resilience of Populus Through Systems Biology Approaches
Sharon L. Doty1* (firstname.lastname@example.org), Andrew W. Sher1, Robert Tournay1, Darshi Banan1, Jayde Aufrecht2, Sun Woo Chung1, Jun Hyuk Jeon1, Matt Hendrickson1, Sriram Parasurama1, Morgan Raimondo1, Emma Gomez-Rivas1, Amir Ahkami2, Adam Deutschbauer3, and Soo-Hyung Kim1
1University of Washington; 2Pacific Northwest National Laboratory; and 3Lawrence Berkeley National Laboratory
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 (multiomics) data from greenhouse and field experiments will allow development of 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.
Poplar trees are important feedstocks for bioenergy and ecosystem services, but more efficient and resilient growth is essential for sustainability. The microbiome of wild poplar is a rich resource for plant growth promotion with some of the contributing micro-organisms able to provide nitrogen and bioavailable phosphorus. In addition, these micro-organisms may promote plant tolerance of other environmental stresses as well, such as drought. The first objective of this project is to unravel the molecular mechanisms of nitrogen fixation in an optimized constructed community of endophytes isolated from wild poplar. Initially studying a single aerobic diazotrophic strain with highly dynamic nitrogen-fixation, researchers characterized N-fixation using a nitrogenase gene promoter fusion to green fluorescent protein, fluorescence-activated cell sorting, poplar rhizo-chip assays, and a time series of 15N-guided molecular analyses. Nitrogenase gene expression patterns both in vitro and in planta suggested environmental signals are at play, and the resulting signature of nitrogenous compounds was identified using 15N-metabolomics.
A synergistic effect of specific non-diazotrophic strains with diazotrophs was revealed in vitro, moving towards an understanding of inter-species cooperation in a constructed community. Identification of the potential crosstalk molecules between strains will be a next step. A constructed community of eight endophyte strains was optimized with complementary symbiotic traits including nitrogen fixation and synergistic interactions, phosphate solubilization, and hormone production. Genome-scale transposon mutagenesis of two of the diazotrophs of the constructed community is complete, allowing for a series of experiments to be conducted to evaluate the genetic requirements for nitrogen fixation by these aerobic strains. Directed mutagenesis of specific genes is underway. To determine the molecular and physiological impacts of N-fixing endophytes on the host plant, the team is conducting both field and greenhouse level studies. In April 2022, a field site at the Roza Research Station in Prosser, Washington, was planted with poplar trees inoculated with the constructed community. Trees are being monitored and samples collected for analysis of the impacts of the endophytes versus controls at multiple levels including plant physiological, genomic, and metabolomics levels. Meanwhile, a series of more controlled greenhouse level experiments have been performed, with more currently in progress.
Full genomic sequencing and de novo assemblies were completed for the strains making up the constructed community. The annotated draft-assemblies were submitted to the Type (Strain) Genome Server (TYGS) for whole genome-based taxonomic classification, with four strains (Azospirillum sp. SherDot2, Sphingobium sp. WW5, Herbiconiux sp. 11R-B1, and Rhizobium sp. PTD1) classified as potentially novel species and four strains identified to the species level (Rahnella aceris R10, R. aceris WP5, Azotobacter beijerinckii SherDot1, and Rhodotorula graminis WP1). None of the strains were predicted to be human pathogens by PathogenFinder (v1.1), and in silico analyses were completed cataloging the genetic features associated with plant growth–promoting (PGP) traits for each of the strains. Strain-specific-primer (SSP) sets have been designed and are currently being screened for the ability to successfully target unique DNA sequences in each of the strains. These will be used in digital droplet PCR assays to verify colonization and localization by plant compartment and to estimate the relative abundance of the strains within and between treatment groups. The SSPs will be used in conjunction with metabarcoding studies targeting the 16S rRNA genes to investigate changes in the community structure of the plant microbiome from the field and greenhouse experiments under abiotic stresses, providing information toward the objective of identifying the mechanisms of plant impacts on the microbial community.
By studying both the impacts of a constructed microbial community on the host plant as well as the impacts of the plant on the microbiome, the team hopes 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.
This research was supported by the DOE Office of Science, Biological and Environmental Research (BER) Program, grant no. DE-SC0021137.