High-throughput rhizosphere biosensor development
EMSL Project ID
60124
Abstract
The development of robust quantitative in situ bacterial biosensors that report on inter-organismal rhizosphere signaling, soil conditions, and plant health remains a pivotal goal for the research community. EMSL is uniquely poised to provide user resources that make this goal achievable. This project will result in a pipeline that users can request for the development of custom biosensors, as well as a platform for biosensor benchmarking. The choice of plant, stress condition, chemical target, or host microbe for biosensor development will ultimately be driven by EMSL users, and EMSL must therefore generate adaptable integrated research platforms that facilitate biosensor development for a wide range of user needs. Regardless of user-specific needs, a critical obstacle preventing rhizosphere biosensor development is the lack of genetic components that report predictably to specific plant or rhizobacteria signals. To meet this need, we propose to develop an EMSL user capability that accelerates the discovery, optimization, and validation of rhizosphere biosensors. Our research objectives are to [1] perform high-throughput discovery and optimization of genetic components for microbial biosensors, [2] simplify/accelerate the identification of condition-specific root exudate metabolites that can serve as rhizosphere-localized biomarkers of plant health and biosensor input, and [3] develop a novel benchtop plant-microbe cultivation platform for biosensor validation. As a proof-of-concept, our initial efforts will focus on the plant growth-promoting bacteria Pseudomonas fluorescens SBW25, for which we have developed sophisticated genomic tools, and its interactions with the bioenergy feedstock crop Sorghum bicolor grown under N-deficient conditions. Additionally, because a prospective EMSL user may desire a biosensor that responds to a specific metabolite, we will also generate a biosensor that quantitatively reports the abundance of the known and agriculturally-relevant S. bicolor root exudate methyl 3-(4-hydroxyphenyl)propionate (MHPP). Our high-throughput biosensor discovery and optimization platform will build on a multi-omics pipeline recently developed at PNNL (LDRD, Elmore). This pipeline uses a combination of differential gene expression data, candidate promoter engineering, high-throughput functional genomics, and FACS at EMSL for high-throughput biosensor development. Together, these data guide the design of synthetic gene cassettes that function in heterologous bacterial hosts. To develop and test biosensors over time, we will also develop a novel benchtop research platform that provides unique advantages for studying plant-microbe metabolite exchange. Our designed platform allows cultivation of physically separated plants and microbes while allowing plant-microbe signaling and cross-feeding through a shared exometabolite pool. Such a system allows researchers to monitor biosensor output and other microbial parameters throughout plant development without destructively sampling the plant host. We will validate biosensors developed here by growing them within the novel platform and in the presence of sorghum (+/- stress) across plant development. In an unfunded collaboration with Professors Carrie Masiello & Joff Silberg of Rice University (not described in the proposal), we will also onboard and test published biosensors for sorghum root exudates (e.g. naringenin) coupled to fluorescence and VOC reporters compared to our novel SBW25 biosensors with sorghum plants in soil.
Project Details
Start Date
2021-10-01
End Date
N/A
Status
Active
Released Data Link
Team
Principal Investigator
Co-Investigator(s)
Team Members