UNDERSTANDING PLANT ROOT BORDER CELLS AND THEIR SECRETIONS IN MEDIATING RHIZOSPHERE MICROBIOME DYNAMICS

Overarching GoalThe overarching goals of this project are to form the foundation of border cell secretory activities in rhizosphere manipulation and develop multidisciplinary skills for the post-doctoral fellow.Project ObjectivesTrainingTheoretical, hands-on operational learning, and applications of Trapped Ion Mobility Spectroscopy (TIMs) coupled to a hybrid quadrupole time-of-flight mass spectrometer (QToF-MS) in nontargeted metabolomics experiments.Milestones for training objective one include:Receiving training from Bruker personnel in proper operation and management of the Sumner lab TIMS-QToF instrument.Analysis of border cell secretions using UHPLC-TIMS-QToF-MS.Utilization of border cell secretion TIMS-QToF data to identify secreted metabolites and derive targets for bioactivity assays.Application of qRT-PCR to Medicago truncatula root border cells.Milestones for training objective two include:Reaching out to collaborators on campus for instructions in proper qRT-PCR usage and methods development.Conduction of experimental protocols we develop for specific application to border cells.Working with our collaborators on proper protocols for data publication.Probing for new proteins in Medicago truncatula root border cells using immunoblots.Milestones for training objective three include:Collaboration with immunoblot experts on MU campus to learn proper techniques for performing immunoblots.Designing new protocols to make border cells suitable for immunoblot analyses.Performing preliminary immunoblot analyses utilizing a common cytosolic marker as a proof-of-concept study.Collaborating with other researchers on campus to design antibodies (if necessary) or order antibodies to probe candidate proteins associated with specialized metabolite secretion.Collaborating with other laboratories to learn proper data presentation methods.Employment of Bioactive Metabolites in Bacterial Growth Inhibition Assays.Milestones for training objective four include:Performing background research on proper materials/methods for conducting growth inhibition assays (protocols for these assays are readily available).Conducting the growth inhibition assays, optimize, and observe the results.Collaboration and literature gathering to learn proper publication methods.ResearchEstablishment and identification of secreted metabolites arising specifically from isolated border cells of Medicago truncatula under non-elicted (baseline) and microbial elicted conditions using Ultra High Performance Liquid Chromatography (UHPLC) coupled to Trapped Ion Mobility Spectrometry-Time of Flight-Mass Spectrometry (TIMs-ToF-MS). Ultimately this will fill the knowledge gap of border cell specific contributions to rhizosphere chemistry.Milestones for research objective one include:Development of a protocol for collection of border cell secreted metabolites.Analysis of secreted compounds by UHPLC-TIMS-ToF-MS.Identification of newly secreted metabolites for the purpose of this objective and for later bioactivity studies in objective three.Obtainment (via commercial vendors) or in-house purification of newly secreted metabolites for the purpose of this objective and bioactivity studies in objectiveEstablishment of the mechanisms of specialized metabolite secretion by probing border cell extracts for changing ABC and MATE transporters associated with movement of specialized metabolites utilizing Quantitative Reverse Transcription- Polymerase Chain Reaction (qRT-PCR) and Immunoblot analysis. This will ultimately establish border cell mechanisms of specialized metabolite secretion.Milestones for research objective two include:Development of a protocol for collection of border cell RNA and execution of qRT-PCR experiments.Execution of qRT-PCR experiments.Development of a protocol for extracting proteins from border cells.Design or purchase of antibodies targeting specific transporters (including trial/error in antibody design).Execution ofimmunoblot experiments.Establishment of secreted specialized metabolite roles in relation to Sinorhizobium meliloti (a symbiont of Medicago truncatula) and Pseudomonas syringae pv. tomato DC3000 (a pathogen known to infect Medicago truncatula) using growth inhibition assays. This will ultimately provide insight into the roles of specific phytochemicals in plant defense and symbiosis.Milestones for research objective three include:Acquiring Sinorhizobium meliloti and Pseudomonas syringae pv. DC3000.Culturing and storage of glycerol stocks the aforementioned bacteria using well established medias and methodologies.Culturing of bacteria for growth inhibition assays.Completion of growth inhibitions assays using wide-range dilutions of a minimum of three pure specialized metabolites from research objective one.