This article describes detailed protocols for ecosystem fabrication of devices (EcoFABs) that enable the studies of plants and plant-microbe interactions in highly controlled laboratory conditions.
Beneficial plant-microbe interactions offer a sustainable biological solution with the potential to boost low-input food and bioenergy production. A better mechanistic understanding of these complex plant-microbe interactions will be crucial to improving plant production as well as performing basic ecological studies investigating plant-soil-microbe interactions. Here, a detailed description for ecosystem fabrication is presented, using widely available 3D printing technologies, to create controlled laboratory habitats (EcoFABs) for mechanistic studies of plant-microbe interactions within specific environmental conditions. Two sizes of EcoFABs are described that are suited for the investigation of microbial interactions with various plant species, including Arabidopsis thaliana, Brachypodium distachyon, and Panicum virgatum. These flow-through devices allow for controlled manipulation and sampling of root microbiomes, root chemistry as well as imaging of root morphology and microbial localization. This protocol includes the details for maintaining sterile conditions inside EcoFABs and mounting independent LED light systems onto EcoFABs. Detailed methods for addition of different forms of media, including soils, sand, and liquid growth media coupled to the characterization of these systems using imaging and metabolomics are described. Together, these systems enable dynamic and detailed investigation of plant and plant-microbial consortia including the manipulation of microbiome composition (including mutants), the monitoring of plant growth, root morphology, exudate composition, and microbial localization under controlled environmental conditions. We anticipate that these detailed protocols will serve as an important starting point for other researchers, ideally helping create standardized experimental systems for investigating plant-microbe interactions.
The application of beneficial plant microbes in agriculture offers great potential to increase sustainable food and biofuel production to provide for a growing population1,2,3,4. A significant amount of work supports the importance of plant microbiomes in plant nutrient uptake, tolerance to stresses, and resistance to disease5,6,7,8. However, it is difficult to investigate these mechanisms of plant-microbe interactions in field ecosystems due to the complexity and associated irreproducibility and inability to precisely control microbiome composition and genetics (e.g., using microbial mutants)4,9,10.
One strategy is to construct simplified model ecosystems to enable controlled, replicated laboratory experiments investigating plant-microbe interactions to generate insights that can be further tested in the field10,11,12. This concept builds on traditional approaches using plants grown in soil-filled pots or on agar slabs within greenhouses or incubators13. Although these will likely remain the most widely used approaches, they lack the ability to precisely monitor and manipulate plant growth environments. To these ends, rhizoboxes and rhizotrons represent a major improvement in the ability to study below-ground processes14,15, and, first protocols were published for analyzing rhizosphere metabolites in soil16. More recently, to enable high throughput analysis, advanced microfluidic devices13,17 such as Plant Chip18,19, RootArray20, and RootChip21, have been developed as efficient tools for plant phenotyping with micrometer-scale spatial resolution to monitor the early growth stages of the small model plant Arabidopsis thaliana in liquid flow medium. Recently, a two-layer imaging platform was described that enables root hair imaging of Arabidopsis thaliana at seedling stage with a microfluidic platform22.
Here, detailed protocols for constructing controlled laboratory devices (EcoFABs) are provided, for studying plant microbe interactions and show that they can be used to study diverse plants including Arabidopsis thaliana, Brachypodium distachyon23, the ecologically important wild oat Avena barbata, and the bioenergy crop Panicum virgatum (switchgrass). EcoFAB is a sterile plant growth platform that includes two primary components: the EcoFAB device and sterile plant-sized transparent container. The EcoFAB device is made from a polydimethylsiloxane (PDMS) manufacturing process that involves casting PDMS layers from a 3D printed plastic mold and bonding PDMS layers onto microscope slides using methods previously reported24,25. The detailed procedures of EcoFAB workflow, such as device fabrication, sterilization, seed germination, seedling transplantation, microbe inoculation/cocultivation, sample preparation, and analysis, are described in this protocol (Figure 1). Further modifications of the basic workflow are described, including the installation of computer controlled LED grow lights and the utilization of solid substrates. The utilization of imaging techniques to investigate root morphology changes, microbial colonization of roots, and mass spectroscopic imaging of root exudates are described. We anticipate that the simple, inexpensive design based on readily available materials, as well as the detailed protocols presented here, will turn the EcoFAB platform into a community resource, standardizing laboratory plant-microbiome studies.
The protocols reported here for using ecosystem fabrication to create EcoFABs provides community resources for systematic plant biology studies in highly controlled laboratory conditions. Advances in 3D printing provide widely accessible technologies for constructing and iteratively refining EcoFAB designs. The root chamber presented here is found to be well suited for imaging microscopy and maintaining sterility, enabling controlled addition of microbes to investigate plant-microbe interactions.The EcoFAB platform is compatible with various plant species. It is important to recognize physiological effects of growing the plants within the narrow root chamber such that additional experiments will be required to generalize findings to plants growing in natural environments.
The use of sterile chambers and LED grow light enables the investigation of the effects of various light conditions, including wavelength, intensity, and duration, on plant growth and related physiological parameters in parallel. Reversible bonding root chambers allow the use of solid substrates as well as to spatially collect solid samples for biochemical and genetic analysis. The applications of solid substrates, such as soils, sand, and quartz beads, offer the possibilities of using EcoFABs to construct more ecologically relevant laboratory ecosystems. However, all the systems presented here use saturated liquid (hydroponic cultures) which are not an accurate reflection of most soils and it will be important to further refine these designs to maintain air pockets within the soil such that they better represent natural soils.
The use of both simple cameras and microscopes is described to image root system morphology development at both bulk to cellular levels. This suitability for monitoring root morphology imaging and quantification will likely be helpful for understanding the regulatory mechanisms of plant physiological and molecular signals triggered by plant genotypic adaptions to growth conditions. However, a limitation for studying physiological root development is the current horizontal placement of the EcoFAB device. In natural environments, the roots gravitropic response leads to a predominantly vertical development of the root system. Thus, the horizontal system presented here likely differs in some factors from a natural environment, and the fabrication of EcoFAB systems with vertical placement of the root chamber is a desirable goal for future EcoFAB versions. Although the current EcoFAB devices are placed horizontally, the analysis of root morphology parameters in various conditions, or in response to microbes, is possible. High resolution imaging can be applied to capture root colonization dynamics of single isolates or communities, providing information about which plant parts are colonized in various nutrient sufficient and deficient conditions. It is anticipated that such studies will provide important new insights into how plant microbiomes are assembled, and how these dynamics change over time, for example as the roots develop.
Microfluidic devices enable imaging of very young plants, and usually the amount of metabolites collected is not sufficient for LCMS analysis. Soil-based systems, such as rhizotrons, allow the imaging of root morphology when either the plants are transformed with chemiluminescent construct (Glo-root) or with NMR-based methods33,34. Metabolite extractions from these systems are time consuming because of large volume of samples. EcoFABs are a combination of both: the fabrication is similar to microfluidic devices. EcoFABs were designed to be simple and inexpensive to reproduce, but the size of the chamber can be adjusted to grow plants with small or large root systems, up to their reproductive stages. Simultaneous observations of root morphology changes and root exudation are possible. The system is sterile, enabling controlled addition of specific microbes.
EcoFABs are designed to enable controlled introduction and sampling of microbes and metabolites. Specifically, samples collected from root growth chambers are found to be sufficient for mass spectroscopic metabolite profiling. The integration of mass spectrometry imaging (e.g., NIMS technique presented here) provides a non-destructive approach of studying metabolite spatial distributions of root systems. This technique will likely be helpful in future stable isotope tracing experiments and mapping microbial localization to specific metabolites36. While this protocol has focused on single isolates, the same design can certainly be used for more complex communities. The sample volumes and biomass within the EcoFABs are likely more than sufficient for further integration with DNA sequencing technologies, which will be important to characterizing and monitoring microbial community structure and gene expression.
In conclusion, this protocol details the fabrication of laboratory ecosystems designed for the investigation of plant-microbe interactions, with emphasis on simple and accessible methods that can easily be implemented and extended by researchers around the world. Current efforts are aimed at demonstrating the reproducibility between labs and the integration of a temperature control system such that each EcoFAB will have independently controlled light and temperature. A further advancement of the system will be the integration of automated sampling and refilling of the EcoFAB root chambers and the development of reproducible protocols for establishing relevant plant microbiomes within EcoFABs.
The authors have nothing to disclose.
This work was supported by Laboratory Directed Research and Development (LDRD) program of Lawrence Berkeley National Laboratory supported by the Office of Science, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231 and an award DE-SC0014079 from the U.S. Department of Energy Office of Science to UC Berkeley. Work at the Molecular Foundry was supported under U.S. Department of Energy Contract No. DE-AC02-05CH11231. We also thank Suzanne M. Kosina, Katherine Louie, Benjamin P. Bowen, and Benjamin J. Cole at Lawrence Berkeley National Laboratory for all their help.
3D printed custom mold | LBNL | STL files available here www.eco-fab.org; The EcoFABs molds described here were printed by FATHOM: http://studiofathom.com | |
Dow sylgard 184 silicone elastomer clear kit | Ellsworth Adhesives | 184 SIL ELAST KIT 0.5KG | |
Air duster spray | VWR | 75780-350 | any compressed gas duster should work |
15 gauge blunt needle | VWR | 89166-240 | |
5 mL syringe with Luer-Lok Tip | VWR | BD309646 | |
3”x2” microscope glass slide | VWR | 48382-179 | |
1.75" x 2.56" x 3.56" EcoFAB box | Amazon | B005GAQ25Q | |
4” x 3 ¼” microscope glass slide | Ted Pella | 260231 | |
4.87" x 4.87" x 5.50" EcoFAB box | Amazon | B00P9QVOS2 | |
Plasma Cleaner | Harrick Plasma | PDC-001 | |
3D printed custom clamp | LBNL | STL files available from Trent Northen's lab | |
Sterile hood | AirClean Systems | AC600 Series PCR Workstations | |
PTFE syringe tubing | Sigma-Aldrich | Z117315-1EA | |
Ethanol | VWR | 89125-172 | |
Bleach | |||
Murashige and Skoog (MS) Macronutrient Salt Base | Phytotechnologies Laboratories | M502 | |
Murashige and Skoog (MS) Micronutrient Salt Base | Phytotechnologies Laboratories | M554 | |
Soil | Hummert International | Pro-Mix PGX | |
Phytagel | Sigma-Aldrich | 71010-52-1 | |
Arabidopsis thaliana | Lehle Seeds | WT-24 Col-4 Columbia wild type | |
Brachypodium distachyon | LBNL | Standard Bd-21 line | Available from John Vogel's lab |
Panicum virgatum | The Samuel Roberts Noble Foundation | Alamo switchgrass | |
Micropore tape | VWR | 56222-182 | |
LC-MS grade methanol | VWR | JT9830-3 | |
Lyophilizer | LABCONCO | FreeZone 2.5 Plus | |
SpeedVAC concentrator | Thermo Scientific | Savant™ SPD111 SpeedVac | |
Ultrafree-MC GV Centrifugal Filter-0.22 µm | Millipore | UFC30GV00 | |
Liquid chromotography system | Agilent | Agilent 1290 LC system | |
Q Exactive mass spectrometer | Thermo Scientific | Q Exactive™ Hybrid Quadrupole-Orbitrap MS | |
NIMS chip and custom MALDI plate | LBNL | For detailed protocol see: doi:10.1038/nprot.2008.110 | |
MALDI mass spectrometer | AB Sciex | TOF/TOF 5800 MALDI MS | |
Nano-coated LED grow light strip | LED World Lighting | HH-SRB60F010-2835 | |
Power supply | LED World Lighting | MD45W24VA, LV100-24N-UNV-J | |
TC420 controller | Amazon | B0197U7R8Q | |
Silicone LED clips | Amazon | B00N9X1GI0 | |
Hot glue gun | Amazon | B006IY359K | |
Female-to-bare LED connector cable | LED World Lighting | HH-F05 | |
Female-to-male LED connector extension cable | LED World Lighting | HH-MF1 | |
20AWG 2-wire cable | LED World Lighting | 6102051TFT4 | |
WAGO 221-415 Splicing Connector | LED World Lighting | 221-415 |