Name: a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment
Uploaded: 2010-08-27T17:30:00
Description: Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

Our work is supported by grants from NIH/NIGMS, NSF, USDA/NIFA, and BARD to VC.

Background
Symplastic transport of macromolecules through plant intercellular connections, the plasmodesmata, is of interest to many plant pathologists and biologists. For example, several viral proteins are known to regulate plasmodesmal size exclusion limits to enable viral movement1-3. Also, some endogenous proteins, among them important developmental regulators, are assumed to move from cell to cell, presumably through plasmodesmata, to function non-cell-autonomously4....

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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,...

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<td>Gold microparticles, 1.0 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
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<td>Gold microparticles, 0.6 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2263</td>
<td>&nbsp;</td>
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<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
<td>&nbsp;</td>
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<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
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<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2432</td>
<td>&nbsp;</td>
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a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

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).

Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

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.

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

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 ...

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