Single-Cell Characterization of Microbiomes via Split-Pool Ligation Transcriptomics
EMSL Project ID
60119
Abstract
While single-cell technologies are rapidly becoming a standard for measuring expression in eukaryotic organisms, their utility for prokaryotes is facing substantial technical barriers including smaller cell size and greater genetic heterogeneity. To address these challenges, the proposed research seeks: (i) to establish a high-throughput split-pool ligation transcriptomics (micro-SPLiT RNAseq) approach to detect microenvironment-specific variations in mRNA abundances within microbial subpopulations and (ii) to integrate single-cell transcriptomics analysis with fluorescent in situ hybridization (FISH)-based workflow for high-resolution in vivo localization of individual microbial activities within populations. The utility of our approach will be tested for characterization of endophytic functions and interactions conferring drought tolerance in bioenergy crops.
The proposal directly addresses DigiPhen research priorities related to the Single Cell Biology and HTP Omics areas. A successful outcome will significantly enhance EMSL technical capabilities in high-throughput detection of gene expression heterogeneities within microbial populations facilitating discovery of novel functions and regulatory mechanisms. It will also pave way towards increasing the resolution on omics analyses to reveal unique cell-specific phenotypes and their genetic drivers as they relate to the behavior of cell populations, microbial communities, and host-microbe systems. This, in turn, will lead to outcomes that are of interest to EMSL sponsors and user community through enabling high-throughput multimodal phenotyping of plants and microbes to enable the design and engineering of biological systems for energy or environmental applications.
Our research plan addresses overarching technical challenges related to microbial single-cell measurements through a stage-gated strategy that employs recently developed transcriptomics methodology and well-established imaging and plant biology techniques. Our activities are organized around three synergistic tasks. First, we will apply micro-SPLiT transcript barcoding approach2 to enable single-cell measurements of mRNA abundances within simplified (2-4 member) model consortia to establish the sensitivity and resolution baselines. Second, we will explore the utility of upstream non-destructive cell separation, which will be optimized for compatibility with the micro-SPLIT barcoding workflow, to enable analysis of complex microbial systems including host-microbe communities. Finally, we will extend our activities to employ micro-SPLiT RNAseq to plant-microbe interactions to test and analyze outcomes of bioaugmentation strategies that are used to enhance plant resilience. This activity will not only be used for proof-of-concept but also to identify a set of differentially expressed genes by micro-SPLiT RNAseq to design transcript-specific FISH probes. In vivo characterization through high-resolution imaging across a range of temporally resolved environmental conditions will be carried out to infer putative mechanisms of plant-endophyte interactions within specific parts of the plant.
Achieving the goals of the proposal is a formidable task, but a strong foundation for success has been laid down through research accomplishments and technical capabilities of the assembled technical team. First, we leverage the existing strengths of the assembled team in systems microbiology, high throughput transcriptomics of multi-species systems, and functional genomics (Beliaev, McClure, Markillie), plant physiology (Ahkami, Mundree) and augmented them with state-of-the-art technical capabilities in high-resolution cell separation and imaging (Hu, Chrisler). Second, our research plan that emphasizes the long-term goal of understanding activities of endophytic microorganisms beneficial to bioenergy crops addresses specific technical challenges related to microbial single-cell measurements through a stage-gated strategy that employs well-established analytical methodology and tested plant biology techniques. Third, we are extending our studies to network analysis of global gene expression of mRNA and protein activity that can provide experimentally testable predictions of plant-microbe interactions and inform novel bioaugmentation strategies to enhance plant resilience.
Project Details
Start Date
2021-11-16
End Date
2023-12-31
Status
Closed
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members