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

Ons werk wordt ondersteund door subsidies van NIH / NIGMS, NSF, USDA / NIFA, en BARD naar VC.

Achtergrond Symplastic transport van macromoleculen door de plant intercellulaire verbindingen, de plasmodesmata, is van belang voor veel plant-pathologen en biologen. Bijvoorbeeld, zijn verschillende virale eiwitten waarvan bekend is dat plasmodesmal size exclusion grenzen te reguleren, zodat virale beweging 1-3. Ook zijn sommige endogene proteïnen, waaronder belangrijke ontwikkeling toezichthouders, aangenomen om van cel naar cel, vermoedelijk door plasmodesmata, tot niet-cel-aut...

<ol>
	<li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+non-destructive+cell-to-cell+and+systemic+movement+of+plant+viruses.+.">The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses. .</a> Crit Rev Plant Sci. 23, 195-250 (2004).</li><li>Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viral+movement+proteins:+agents+for+cell-to-cell+trafficking+of+viral+genomes.">Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes.</a> Virology. 344, 169-184 (2006).</li><li>Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+spread+by+diffusion+on+ER-associated+movement-protein-rafts+through+plasmodesmata+gated+by+viral+induced+host+beta-1,3-glucanases.">Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host beta-1,3-glucanases.</a> Semin Cell Dev Biol. 20, 1074-1081 (2009).</li><li>Zambryski, P. C., Crawford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata:+gatekeepers+for+cell-to-cell+transport+of+developmental+signals+in+plants.">Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants.</a> Annu Rev Cell Dev Biol. 16, 393-421 (2000).</li><li>Rivero-Lepinckas, L., Crist, D., Scholl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+plants+and+presercation+of+seeds.">Growth of plants and presercation of seeds.</a> Methods Mol Biol. 323, 3-12 (2006).</li><li>Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transient+genetic+transformation+of+Arabidopsis+leaves+by+biolistic+bombardment.">Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment.</a> Nat Protoc. 4, 71-77 (2009).</li><li>Oparka, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+but+not+branched,+plasmodesmata+allow+the+nonspecific+trafficking+of+proteins+in+developing+tobacco+leaves.">Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.</a> Cell. 97, 743-754 (1999).</li><li>Imlau, A., Truernit, E., Sauer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+and+long-distance+trafficking+of+the+green+fluorescent+protein+in+the+phloem+and+symplastic+unloading+of+the+protein+into+sink+tissues.">Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.</a> Plant Cell. 11, 309-322 (1999).</li><li>Gisel, A., Barella, S., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+regulation+of+symplastic+trafficking+during+development+in+Arabidopsis+thaliana+apices.">Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices.</a> Development. 126, 1879-1889 (1999).</li><li>Kim, I., Cho, E., Crawford, K. M., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+movement+of+GFP+during+embryogenesis+and+early+seedling+development+in+Arabidopsis.">Cell-to-cell movement of GFP during embryogenesis and early seedling development in Arabidopsis.</a> Proc Natl Acad Sci USA. 102, 2227-2231 (2005).</li><li>Crawford, K. M., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-targeted+and+targeted+protein+movement+through+plasmodesmata+in+leaves+in+different+developmental+and+physiological+states.">Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states.</a> Plant Physiol. 125, 1802-1812 (2001).</li><li>Roberts, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+unloading+in+sink+leaves+of+Nicotiana+benthamiana:+comparison+of+a+fluorescent+solute+with+a+fluorescent+virus.">Phloem unloading in sink leaves of Nicotiana benthamiana: comparison of a fluorescent solute with a fluorescent virus.</a> Plant Cell. 9, 1381-1396 (1997).</li><li>Guenoune-Gelbart, D., Elbaum, M., Sagi, G., Levy, A., Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+(TMV)+replicase+and+movement+protein+function+synergistically+in+facilitating+TMV+spread+by+lateral+diffusion+in+the+plasmodesmal+desmotubule+of+Nicotiana+benthamiana.">Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana.</a> Mol Plant Microbe Interact. 21, 335-345 (2008).</li></ol>

De sleutel voor het succes van de symplastic transport-test is het verkrijgen van een hoge efficiëntie van de omzetting, die de productie van statistisch significante en gemakkelijk waarneembaar signaal clusters mogelijk maakt. Dit kan worden bereikt met behulp van de bladeren geoogst van gezonde, robuuste planten, en het voorbereiden van gouddeeltjes bekleed met een zuivere en geconcentreerde DNA voorbereiding. Met behulp van de bladeren in dezelfde groeifase is ook van vitaal belang voor ...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Gold microparticles, 1.0 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
<tr>
<td>Gold microparticles, 0.6 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2263</td>
<td>&nbsp;</td>
<tr>
<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
<td>&nbsp;</td>
<tr>
<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
<td>&nbsp;</td>
<tr>
<td>Helios cartridge preparatory station</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2420</td>
<td>&nbsp;</td>
<tr>
<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
<td>&nbsp;</td>
<tr>
<td>Helios gene gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2432</td>
<td>&nbsp;</td>
<tr>
<td>Helium gas regulator</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2413</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

Hier presenteren wij een eenvoudige en snelle protocol op te sporen en te beoordelen van de mate van cel-cel macromoleculaire transport in planta. In dit protocol is een fluorescent gelabeld eiwit van belang tijdelijk, uitgedrukt in plantenweefsel na biolistische levering van haar coderend DNA te construeren. De intra-en intercellulaire verdeling van de gelabelde eiwit wordt vervolgens geanalyseerd door confocale microscopie. We beschrijven deze technologie in detail, het verstrekken van stap-voor-stap protocollen test en evaluatie van de mate van symplastic eiwit transport in drie plantensoorten, Arabidopsis thaliana, Nicotiana benthamiana en N. tabacum (tabak).

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

Macromoleculaire de handel tussen de plantencellen kan worden beoordeeld door kortstondig te drukken een fluorescent-gelabeld eiwit van belang en de analyse van de intra-en intercellulaire distributie door confocale microscopie.

Een cel-cel Macromoleculaire Transport Assay In Planta Door gebruik te maken biolistische Bombardement

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 ...

Research

JoVE Journal

Biology

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

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

De voorbereiding van Gene Gun Bullets and biolistische Transfectie van Neuronen in Slice Cultuur

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Venster op een Microworld: Eenvoudig Microfluïdische Systemen voor het bestuderen van microbiële transport in poreuze media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Generation of Transgenic C. elegans by Biolistic Transformation

Generation of Transgenic C. elegans by Biolistic Transformation

Generatie van transgene C. elegans Door biolistische Transformatie

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

Oogst en Cryo-koeling kristallen van membraaneiwitten Grown in lipide mesofasen voor structuurbepaling door Macromoleculaire Kristallografie

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

In vitro analyse van PDZ-afhankelijke CFTR Macromoleculaire signaalcomplexen

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

Kweken<em&gt; Caenorhabditis elegans</em &gt; In Axenische Liquid Media en Creatie van transgene Worms door bombardement met microdeeltjes

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

Open Source High Content Analysis Door gebruik te maken van de automaten Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

Methoden om te studeren epitheliale Transport Protein functie en de expressie in Inheemse darm en Caco-2-cellen gekweekt in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

Kraaktemporale parameters van cellulaire exocytose in micropatterned cellen kwantificeren

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

Optimalisatie van kristalgroei voor neutronenmacromoleculaire kristallografie

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...