Location: Plant Gene Expression Center
Project Number: 2030-12210-002-005-A
Project Type: Cooperative Agreement
Start Date: Oct 1, 2019
End Date: Feb 14, 2023
The goal of this project is to investigate the molecular causes of a recently described Actinobacterial enrichment in the root microbiome of crop plants under drought stress, from the microbial and host perspectives, and the phenotypic consequences of this enrichment for the host plant. The team will use a combination of new genomic technologies and microbiome manipulation strategies, in both lab and field-based experimental designs, using Sorghum bicolor , which represents an excellent agronomic model for drought research and an important feedstock and bioenergy crop. The research can be broadly categorized by the two central objectives of the project: 1) identification of the plant and microbial molecular and genetic pathways that lead to enrichment of Gram-positive bacteria within drought treated roots, and 2) characterization of plant phenotypic response and agricultural yield following inoculation with drought enriched Gram-positive bacterial isolates. The independent but complementary avenues of investigation designed to accomplish these objectives will be conducted in parallel, but will be summarized sequentially below. As the team hypothesizes that the observed enrichment in Gram-positive bacteria is the result of a complex interaction of plant metabolism and microbial physiology, the experiments are designed to identify the genetic basis for drought enrichment from both the plant and microbial perspectives. To address the plant contribution to the enrichment phenomenon, the team will use an untargeted approach that will agnostically probe a diverse array of drought-enriched metabolites; microbial growth assays on a broad range of drought-enriched, plant-derived compounds will be used to test the role of individual metabolites in promoting or deterring the fitness of a large collection of drought-enriched and drought-depleted bacterial isolates.
To identify the role of specific drought-enriched, plant-produced compounds in promoting microbial growth, the Cooperator PI will perform high-throughput phenotyping of isolates selected from preexisting culture collections developed in the ARS PI from the roots of field-grown, drought-stressed sorghum roots. This library currently contains approximately 400 isolates, of which roughly 75% represent drought-enriched lineages, and approximately 15% represent drought-depleted isolates. Isolates were cultured from surface-sterilized roots collected at the peak of drought, using variety of enrichment media including humic acid vitamin/gellan gum, skim milk/gellan gum, and ISP2 agar coupled with antibiotic supplementation, with a particular focus on isolation of Actinobacteria. A subset of approximately 200 of these isolates will be selected for high-throughput growth assays based on the following criteria: 1) abundance patterns within the root microbiome based on previously collected 16S rRNA amplicon datasets (produced as part of EPICON research); 2) similarity to lineages for which closely-related taxa show unconventional abundance patterns; 3) lineages for which growth phenotypes can be readily assayed with our phenotyping platform. This selection process will also take into account data collected as part of an ongoing Community Sequencing Project through the Joint Genome Institute (see ARS PI current support), which is responsible for producing full-length 16S rRNA community profiles on the PacBio Platform from drought-treated sorghum root samples. This data will be used to more precisely map abundance patterns under drought to specific isolates within the collection. Additionally, as part of the CSP a subset of 48 of the drought-enriched sorghum root isolates from the collection will be used to generate full-length isolate genomes on the PacBio platform. Preference in our high-throughput growth assays will be for isolates that have been sequenced (n=18) or which are slated to have been sequenced in the next four months (n=30). To systematically compare the growth phenotypes of bacteria that are enriched and depleted following drought, the Cooperator PI will assay a roughly equal number of bacteria from both classes. In some instances, the Cooperator PI may assay relatives of these bacteria that exist in rhizosphere strain collections (Cooperator PI). The Cooperator PI has developed a high-throughput pipeline for efficient and robust screening of bacterial isolates under a wide range of experimental conditions. In these assays, the Cooperator PI cultivates bacteria under hundreds of growth conditions, including different carbon and nitrogen sources in defined media, as well as their inhibition by stress compounds. In the work, approximately 96 plant produced compounds will be tested against the 200 isolates; these compounds will be selected based on: 1) enrichment within the root metabolome of drought stressed as compared to control treated plants (currently 21 compounds fit this criteria including the aforementioned G3P, as well as ribose, proline, and threonine ); 2) compounds that are hypothesized to be involved in the process.