Location: Plant Gene Expression Center
Project Number: 2030-12210-002-001-N
Project Type: Non-Funded Cooperative Agreement
Start Date: May 19, 2017
End Date: May 18, 2022
We propose to investigate the role of root-associated Actinobacteria in promoting host fitness under drought stress in two plants important to the DOE mission of sustainable biofuels, the JGI Flagship plant, Sorghum bicolor, and Oryza sativa. We have chosen to focus on Actinobacteria because of their ability to survive in extreme environments, their production of many bioactive secondary metabolites, and because preliminary data from our labs suggest that Actinobacteria are reproducibly more abundant in the roots of drought-treated sorghum and rice than in those of well-watered controls. The cause of this enrichment and its implication for plant yield and health are currently unknown. However, many Actinobacteria have been shown to be capable of promoting growth in a variety of plant species, in part through their ability to synthesize a broad range of secondary metabolites recognizable by plants and other microbes. We hypothesize: 1) that the enriched Actinobacteria are actively recruited to the root endosphere in response to shifts in host metabolism, and 2) that they enhance the plant’s drought tolerance either directly, by modulating host transcription, or indirectly, by displacing or suppressing fungal microbes pathogenic to the host plant. To test these hypotheses, four biologists with expertise in sorghum (DCD), rice, (VS), Actinobacteria (MT), and fungi (JT) have designed a series of field and laboratory experiments outlined in this proposal. First, 16S rRNA and ITS2 amplicon sequencing will be used to characterize the bacterial and fungal microbiome of rice and sorghum during drought stress and to guide the selection of specific isolates for downstream genomic and functional characterization. Sequencing of select Actinobacteria will provide information on their genetic properties and metabolic capabilities. Transcriptional profiling of laboratory-grown plant roots inoculated with individual isolates will allow identification of host genetic machinery responsible for sensing and responding to specific microbes. Finally, mass-spectrometry-based metabolomic analysis of both host and microbe(s) will characterize the molecular signals shared between plant and Actinobacteria that allow for the relationship to be established and maintained.
Our project includes four components that will be used to complete the objectives outlined in the abstract above and which are explained in detail in the proposal below: 1) 120 samples each of 16S rRNA and ITS2 amplicon sequencing (2 field conditions x 5 replicates x 3 sample types x 2 time points x 2 for rice and sorghum); ~12 Gb 2) Isolate sequencing for 96 root-associated bacteria from drought-treated roots (48 from sorghum, 48 from rice) and downstream assembly and annotation; ~768 Gb 3) Transcriptome sequencing of 66 samples of gnotobiotically-grown roots following inoculation with drought-enriched endophytes (10 isolates + 1 sterile control x 3 replicates, for both sorghum and rice); ~ 132 Gb 4) LC-MS for 496 samples from roots (2 treatments x 4 replicates, for both sorghum and rice, = 16 samples) and from isolates (10 isolates x 3 treatments x 4 replicates x 2 for polar and non-polar analyses, for both sorghum and rice, = 480 samples). Due to the scope of the proposed work, including the volume and breadth of the proposed experiments, this project requires the involvement of the JGI for library production, sequencing and initial data analysis in order to accomplish the project’s goals.