Source: UTAH STATE UNIVERSITY submitted to
OUTER MEMBRANE VESICLE ACTIVITY IN THE RHIZOSPHERE AND BIOFORTIFICATION APPLICATIONS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
Reporting Frequency
Annual
Accession No.
1009716
Grant No.
(N/A)
Project No.
UTA-01280
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2016
Project End Date
Jun 30, 2021
Grant Year
(N/A)
Project Director
Britt, DA, W.
Recipient Organization
UTAH STATE UNIVERSITY
(N/A)
LOGAN,UT 84322
Performing Department
Biological & Irrigation Engineering
Non Technical Summary
Enhancing agricultural output to accommodate a rapidly growing world population is a top USDA-NIFA priority that is complicated by climate change and variability. Improving agricultural output across the globe will benefit from a bottom-up study of the fundamental role of microbial activity in the root zone where beneficial microbes, i.e. the plant microbiome, play a role analogous to the gut microbiota. The demand for increased agricultural output is further challenged by extremes in growing conditions arising from world-wide climate variability. The effects of stresses on the beneficial soil microbial communities will be investigated here through stress-induced changes in bacterially derived vesicles that can be assayed for stimulation of soil microbes and the host plant. Applications of the isolated and characterized vesicles in plant biofortification represent a unique approach to stimulate crops and enhance output through microbial products.
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
20340992020100%
Goals / Objectives
This project aims to investigate outer membrane vesicles (OMVs) derived from beneficial plant colonizing microbes subjected to a range of stress to understand how vesicle production, properties, and payload vary under these pressures. Four specific goals have been developed and will be conducted over a 5-year research period:1. Develop a root mimetic hollow fiber membrane for culturing beneficial soil microbes as biofilms and isolating OMVs produced under baseline and bacterial stress conditions. (year-1)2. Develop methods to reproducibly generate and analyze bacterial OMVs under baseline and stress conditions. Isolate bacterial OMVs produced by beneficial rhizosphere microbes colonized on a root mimetic hollow fiber microfluidic platform. Analyze isolated bacterial OMVs and screen for distinct OMV properties in response to the stress of engineered nanoparticles (NPs), surfactants, fertilizers, heat, drought, and salt. (years 2-3)3. Assess OMV affinity for biotic surfaces to determine the physical forces influencing vesicle adsorption, assembly, rupture / uptake at interfaces. (years 3-4)4. Assess bioactivity, biofortification, and immune stimulating potential of isolated/characterized OMVs on rhizosphere microbes and host plant. (years 4-5)
Project Methods
For the root-mimetic system commercial ultrafiltration hollow fiber membranes having 185 micron lumen diameters and 30 micron wall thickness and a MW cutoff ~60 kD will be employed. The root-mimetic permits: 1) in-situ fluorescence microscopy imaging to explore the influence of key rhizosphere metabolites (lumen side) and challenges (exterior side) on the architecture of the biofilms, and 2) the isolation of OMVs produced under these challenges without complications from live root cells. Defined media will be introduced into the fiber lumens representing plant root exudate. Temperature, water, and salt stress can be readily implemented along with introduction of fertilizers, surfactants, and nanoparticles to the inter-lumen space.Standard differential centrifugation followed by ultracentrifugation and filtration protocols for OMV isolation will be employed. "In-line" methods for OMV isolation directly from the root mimetic system will be explored by integrating filtration membranes in series with field flow fractionation (FFF) to collect and fractionate OMVs based on size. The OMV content will be assessed using stains and assays specific for protein, nucleic acids, LPS, and small molecule surfactants, signaling molecules, and metabolites. OMV size and charge will be characterized using dynamic light scattering (DLS) and zeta potential, respectively. Size and morphology will be confirmed using high-resolution atomic force microscopy (AFM), which also can be used to probe OMV physiochemical properties such as compliance and adhesion.OMVs may be traced based on autofluorescence if fluorescent metabolites such as phenazines and siderophores are carried by the OMVs. OMV intrinsic fluorescence will be assessed using fluorometry and laser scanning confocal microscopy (LSCM), and distinct OMV fluorescence may again arise depending on the vesicle composition / cargo.Model surfaces in which surface chemistry, roughness, and patterning are strictly controlled will be employed to help distinguish the physical vs. chemical influences on OMV attraction, adsorption (desorption), assembly, and spreading (rupture) at interfaces. Biotic interfaces will be modeled using self-assembly methods to construct lipid monolayers as cell membrane mimics in which phase behavior and formation of domains (e.g. lipid rafts) can be controlled. Real-time OMV adsorption kinetics on surfaces modified to exhibit defined charge, hydrophobicity, roughness, etc. will be conducted using quartz crystal microgravimetry (QCM) and surface plasmon resonance (SPR).The root-mimetic system will be employed to investigate how doses of the characterized OMVs influence the biofilm architecture and properties. In particular, the response of OMV-treated biofilms to the selected stressors will be investigated to determine if OMVs carry signals or act as adjuvants to prime the bacteria to better withstand these challenges.For plant studies OMV treatments will be conducted at multiple stages of plant growth to assess the time of OMV application for biofortification on plant growth and health. Seeds pre-treated with OMVs provide the most direct method for assuring delivery of the vesicles to the rhizosphere during germination. Addition of OMVs to the rhizosphere of plants at later stages of growth will also be explored.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Academic, through professional society presentations and publications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The post-doctoral associate provided the graduate student in-depth training on Raman spectroscopy. The graduate student also received training in density gradient ultracentrifugation. The graduate student presented this work at two conferences focusing on nanotechnology, allowing for interaction with peers and established experts in this field. How have the results been disseminated to communities of interest?Results were presented as a poster at a Gordon Research Seminar (June 2018), a poster at a Gordon Research Conference (June 2018), and a podium presentation at the annual meeting of the Sustainable Nanotechnology Organization (SNO, November 2018). What do you plan to do during the next reporting period to accomplish the goals?The plan of work will focus on linear discriminant analysis of Raman spectra of OMVs collected for control and the abiotic stressor of H2O2 and CuO nanoparticle. These will be compared to Raman for PcO6 cells under the same conditions to identify if selective subset of compounds induced by the stressors in the whole cells are packaged into/on the OMVs. Peak assignments in Raman will be supported by OMV biomolecule quantification, including protein, lipid, and DNA content.

Impacts
What was accomplished under these goals? Outer membrane vesicles (OMVs) were isolated from the beneficial root-colonizing microbe, Pseudomonas chlororaphis O6 (PcO6) under baseline and external stressor conditions. Bacteria where grown as biofilms in the form of bacterial lawns on rich medium (LB) growth plates and subject to hydrogen peroxide (3%) or CuO nanoparticle (30 mg/L) challenges or double distilled water (negative control). Isolation of OMVs from bacterial biofilms is an advancement over the standard protocols using liquid growth (planktonic) conditions. The OMVs were isolated using density gradient ultracentrifugation methods and analyzed with dynamic light scattering and Raman spectroscopy. DLS reported hydrodynamic diameters of OMVs between 150 and 300 nm for all OMV isolates. Raman spectroscopy showed distinct chemical signatures for the OMVs isolated under these different stressors using least discriminate analysis. The Raman for OMVs were contrasted with Raman analysis of whole PcO6 under the same stressor conditions. All OMVs fall within, or just above, the dimensions of nanoparticles (NPs). Linear discriminant analysis shows that exposure to CuO NPs or H2O2 alters the Raman spectra of PcO6 bacteria. This may indicate differences in secretions, including OMVs, from PcO6 cells under these abiotic stressors. The OMVs of PcO6 exposed to CuO NPs or H2O2 show differences in peak intensity of Raman spectra compared to OMVs of PcO6 without stressors. Both PcO6 cells and OMVs show peaks for similar compounds including nucleic acids, amino acids, proteins, lipids and carbohydrates An aromatic carbon peak (3064 cm-1) present in Raman spectra of PcO6 cells is noticeably absent in Raman spectra of OMVs. Differences in intensity of peaks of PcO6 compared to OMVs suggests differences in relative concentrations of compounds.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Presentations Potter, M. K. (Presenter & Author), Bahe, C. (Author Only), Jacobson, A. (Author Only), Anderson, A. J., McLean, J. E. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "CuO nanoparticle influence on wheat photosynthetic efficiency during simulated drought," Washington D.C. (November 14, 2018 - November 17, 2018)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Presentations Potter, M. K. (Presenter & Author), Hanson, C. (Author Only), Anderson, A. J. (Author Only), Vargis, E. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "Nanoparticle influence on bacterial outer membrane vesicles," Washington D.C. (November 14, 2018 - November 17, 2018)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Presentations Cartwright, A. (Presenter & Author), Kunzler, D. (Author Only), Morgan, C. (Author Only), McLean, J. E. (Author Only), Jacobson, A. (Author Only), Anderson, A. J., Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "Protective osmolyte coronal layers to enhance nanoparticle bioavailability and activity," Washington D.C. (November 14, 2018 - November 17, 2018)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Presentations Cartwright, A. (Presenter & Author), McLean, J. E. (Author Only), Anderson, A. J. (Author Only), Jacobson, A. (Author Only), Giasuddin, A. B. M. (Author Only), Kunzler, D. (Author Only), Morgan, C. (Author Only), Valiente, J., Britt, D. W., Gordon Research Conference: Nanoscale science and engineering for agriculture and food, "A novel root mimetic platform for testing the effects of SiO2 nanoparticles on the architecture of beneficial biofilms," GRC, Mount Holyoke, South Hadley, MA. (June 3, 2018 - June 8, 2018)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Presentations Potter, M. (Presenter & Author), Hanson, C. (Author Only), Vargis, E. (Author Only), Britt, D. W., Gordon Research Conference: Nanoscale science and engineering for agriculture and food, "Bacterial outer membrane vesicles: Natures nano," GRC, Mount Holyoke, South Hadley, MA. (June 3, 2018 - June 8, 2018)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Presentations Morgan, C. (Presenter & Author), Cartwright, A. (Author Only), Kunzler, D. (Author Only), Britt, D. W., Gordon Research Seminar: Nanoscale science and engineering for agriculture and food, "Capped nanoparticles improving controlled nutrient delivery to crops," GRC / GRS, Mount Holyoke, South Hadley, MA. (June 3, 2018 - June 4, 2018)


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Academic, through professional society presentations and publications. Changes/Problems:An additional analytical method of Raman spectroscopy will be applied to characterize the isolated OMVs. What opportunities for training and professional development has the project provided?SEM training was completed for a graduate student through the USU Core Microscopy Facility. Nine students from 2-year colleges attending a summer undergraduate research experience in Biological Engineering at USU gained fundamental skills in biological engineering, microbiology, plant growth, and access to advanced analytical equipment, including SEM. How have the results been disseminated to communities of interest?One journal article was published reporting on the development of the root mimetic system and applications. Results were presented as posters at three national conferences. What do you plan to do during the next reporting period to accomplish the goals?Using the developed root mimetic system bacteria biofilms will be assessed for abiotic stress responses, with the aim of inducing OMV production. Standard methods of TFF and centrifugation will be employed for OMVs isolation. Isolation methods will be optimized to provide maximum OMV yields for subsequent characterization.

Impacts
What was accomplished under these goals? Development of an artificial root mimetic using a porous hollow fiber membrane. Determination of biofilm growth as a function of artificial root exudate carbon and nitrogen content up to 1 week. Bacterial biofilm formation assessed using two different plant beneficial microbes. Established biofilms were examined using scanning electron microscopy to assess bacterial response to CuO and ZnO nanoparticle challenges as abiotic stressors. Spherical granules were identified within the PcO6 bacteria comprising the biofilms that had not previously been reported for this microbe. Putative outer membrane vesicles (OMVs) were also identified in the biofilms using SEM.

Publications

  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Potter, M. (Presenter & Author), Doxie, S. (Author Only), McLean, J. E. (Author Only), Anderson, A. J. (Author Only), Jacobson, A. (Author Only), Britt, D. W. (Author Only), Sustainable Nanotechnology Organization, "CuO Nanoparticle Modified Lignification of Wheat," Los Angeles, California. (November 5, 2017 - November 7, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Britt, D. W. (Presenter & Author), Mohammad Giasuddin, A. B. (Author Only), Sustainable Nanotechnology Organization, "Self-assembly of tri-functional and di-functional alkane silanes into hydrophobic silica nanoparticles in aqueous media," Los Angeles, California. (November 5, 2017 - November 7, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Britt, D. W. (Presenter & Author), Potter, M. (Author Only), Anderson, A. J. (Author Only), Villanueva, I. (Author Only), Taylor, T. A. (Author Only), Sustainable Nanotechnology Organization, "Summer education in nano- and biological approaches to protect plants against drought stress," Los Angeles, California. (November 5, 2017 - November 7, 2017)
  • Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Bonebrake, M. (Presenter & Author), Anderson, K. (Author Only), McLean, J. E., Jacobson, A., Anderson, A. J., Britt, D. W., Institute of Biological Engineering, "Characterization of biofilms and metabolites in a synthetic rhizosphere," IBE, Salt Lake City. (March 30, 2017 - April 1, 2017)


Progress 07/01/16 to 09/30/16

Outputs
Target Audience:Target Audience Scientific community. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Opportunities One MS student has been recruited for this project (Michelle Bonebrake) and she has received training from the project director on fundamental microfluidic delivery systems needed for the root-mimetic HFM system. She has also been trained on fluorescence and brightfield microscopy methods used for biofilm characterization. She has developed several prototype root-mimetic platforms. A second MS student (Kaitlyn Anderson) whose research focused on biomedical aspects of HFMs has provided assistance with the root-mimetic development. An undergraduate student volunteer (Matthew Potter) has provided additional assistance by developing methods to potentially create root-mimetics using root epidermal cell sheaths. All students are in the Biological Engineering Department at USU. Training in bacterial culture and plant growth methods to the students was provided by Dr. Anne Anderson in the USU Biology Department. How have the results been disseminated to communities of interest?Dissemination None yet. Dissemination of the preliminary work at the annual Institute of Biological Engineering conference, to be held in SLC, April 1-2, 2017, is planned. What do you plan to do during the next reporting period to accomplish the goals?Plan of Work Using the root-mimetic HFM system the role of outer membrane vesicles (OMVs) as carriers for secondary metabolites will be characterized as a function of rhizoexudate / defined media delivered through the fiber lumens. Fluorescence, brightfield, and scanning electron microscopy methods will be employed to characterize biofilm architectures under baseline and stress response conditions. Methods of controlling the delivery rate of root exudate using gravity feed and capillary action will be investigated as alternatives to syringe pump delivery. Single and mixed component bacterial films of PcO6 and Bs will be investigated for biofilm forming potential and OMV production and cross-communication under baseline and stressed conditions, simulated by different rhizoexudate compositions.

Impacts
What was accomplished under these goals? Accomplishments A root-mimetic model using a hollow fiber membrane (HFM) filter was developed and validated for two root-colonizing bacteria, Pseudomonas chlorororaphis O6 (PcO6) and Bacillus subtilis (Bs), both isolated from wheat. The root-mimetic system has demonstrated robust bacterial biofilm formation in a simulated rhizosphere. The biofilms were fed defined media representing subsets of rhizoexudates, allowing for the assessment of plant components on the biofilm architecture and production of secondary metabolites.

Publications