A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment
Summary
Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.
Abstract
Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).
Protocol
Discussion
The key for the success of the symplastic transport assay is to obtain high transformation efficiency, which allows production of statistically significant and easily detectible signal clusters. This can be achieved using the leaves harvested from healthy, robust plants, and preparing gold particles coated by a pure and concentrated DNA preparation.
Using the leaves at the same growth stage is also vital for the assay reliability. Plasmodesmal aperture is known to be differentially regulated,…
Divulgations
The authors have nothing to disclose.
Acknowledgements
Our work is supported by grants from NIH/NIGMS, NSF, USDA/NIFA, and BARD to VC.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Gold microparticles, 1.0 μm in diameter | Bio-Rad | 165-2262 | ||
| Gold microparticles, 0.6 μm in diameter | Bio-Rad | 165-2263 | ||
| Spermidine | Sigma | S0266-1G | ||
| Tefzel tubing | Bio-Rad | 165-2441 | ||
| Helios cartridge preparatory station | Bio-Rad | 165-2420 | ||
| Tubing cutter | Bio-Rad | 165-2422 | ||
| Helios gene gun | Bio-Rad | 165-2432 | ||
| Helium gas regulator | Bio-Rad | 165-2413 |
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