Name: a &#946;-glucuronidase (gus) based cell death assay
Uploaded: 2011-05-06T15:00:00
Description: Watch this Scientific Journal Video about A Î²-glucuronidase GUS Based Cell Death Assay at JoVE.com

Nicotiana benthamiana plants are grown in a temperature-controlled growth chamber at 25&deg;C . Fully expanded healthy leaves of 3-6 week old plants are used.
Tip: Better results are obtained by using newly emerging leaves
1. Agrobacterium transient infiltration protocol:
Day 1
<ol>
 <li>Streak LB/Rifampicin (25 &mu;g/ml)/Kanamycin (100 &mu;g/ml) agar plates with glycerol stocks of Agrobacterium tumefaciens (strain LBA4404) contai...

<ol>
	<li>Jefferson, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GUS+fusions:+,+&#946;-glucuronidase+as+a+sensitive+and+versatile+gene+fusion+marker+in+higher+plants.">GUS fusions: , &#946;-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.</a> EMBO J. 6, 3901-3901 (1987).</li><li>Nishihara, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+&#946;-Glucuronidase+Gene+in+Pollen+of+Lily+(Lilium+longiflorum),+Tobacco+(Nicotiana+tabacum),+Nicotiana+rustica,+and+Peony+(Paeonia+lactiflora)+by+Particle+Bombardment.">Expression of the &#946;-Glucuronidase Gene in Pollen of Lily (Lilium longiflorum), Tobacco (Nicotiana tabacum), Nicotiana rustica, and Peony (Paeonia lactiflora) by Particle Bombardment.</a> Plant Physiol. 102, 357-357 (1993).</li><li>Hodal, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 87, 115-115 (1992).</li><li>Kabbage, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+BAG+proteins:+a+ubiquitous+family+of+chaperone+regulators.">The BAG proteins: a ubiquitous family of chaperone regulators.</a> Cell Mol. Life Sci. . 65, 1390-1390 (2008).</li><li>Kang, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AtBAG6,+a+novel+calmodulin-binding+protein,+induces+programmed+cell+death+in+yeast+and+plants.">AtBAG6, a novel calmodulin-binding protein, induces programmed cell death in yeast and plants.</a> Cell Death Differ. 13 (1), 84-84 (2006).</li><li>Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Science. 165 (1), 1-1 (2006).</li><li>Takayama, S., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+chaperone+targeting+and+regulation+by+BAG+family+proteins.">Molecular chaperone targeting and regulation by BAG family proteins.</a> Nat Cell Biol. 3 (10), 237-237 (2001).</li><li>Takayama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+functional+analysis+of+BAG-1:+a+novel+Bcl-2-binding+protein+with+anti-cell+death+activity.">Cloning and functional analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell death activity.</a> Cell. 80 (2), 279-279 (1995).</li><li>Vitha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+&#946;-glucuronidase+assay+in+transgenic+plants.">Quantitative &#946;-glucuronidase assay in transgenic plants.</a> Biol. Plant. 35, 151-151 (1993).</li><li>Otha, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+Expression+in+Tobacco+of+a+&#946;-Glucuronidase+(GUS)+Reporter+Gene+Containing+an+Intron+Within+the+Coding+Sequence.">Construction and Expression in Tobacco of a &#946;-Glucuronidase (GUS) Reporter Gene Containing an Intron Within the Coding Sequence.</a> Plant Cell Physiol. 31, 805-805 (1990).</li></ol>

It is often difficult to use cell death detection techniques in plants that are common in mammalian systems. In combination with a GUS reporter system, we present a plant based, sensitive method for the detection and analysis of cell death players. This method takes advantage of the simple fact that live cells are required for GUS expression to occur. To ensure meaningful results and repeatability, it is critical that the cultures harboring the GUS cassette and the gene to be assayed are infiltrated at equal ratios. The...

<table border="1">
	<tbody>
 	<tr>	
 	<th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>	
 	<td>Rifampicin</td>
 <td>VWR</td>
 <td>IC19549001</td>
 <td>25mg/mL stock in DMSO</td>
 </tr>
 <tr>	
 	<td>4'-hydroxy-3',5'-dimethoxyacetophenone (Acetosyringone)</td>
 <td>VWR</td>
 <td>TCD2666</td>
 <td>0.981 g/mL in DMSO (1M stock)</td>
 </tr>
 <tr>	
 	<td>2-(4-morpholino)ethanesulfonic acid monohydrate (MES)</td>
 <td>VWR</td>
 <td>EM-6110</td>
 <td>1.92 g/L in water for making 1 L of infiltration media</td>
 </tr>
 <tr>	
 	<td>Bacto-tryptone</td>
 <td>Fisher</td>
 <td>BP1421-2</td>
 <td>10g/L in water for making 1 L of LB medium</td>
 </tr>
 <tr>	
 	<td>Bacto-yeast extract</td>
 <td>VWR</td>
 <td>EM1.03753.0500</td>
 <td>5g/L in water for making 1 L of LB medium</td>
 </tr>
 <tr>	
 	<td>Sodium chloride</td>
 <td>VWR</td>
 <td>EM-7710</td>
 <td>10g/L in water for making 1 L of LB medium</td>
 </tr>
 <tr>	
 	<td>Sodium phosphate monobasic monohydrate</td>
 <td>VWR</td>
 <td>MK-7868-12</td>
 <td>2.5g/L in water for making 50mM of buffer NaPi</td>
 </tr>
 <tr>	
 	<td>Sodium phosphate dibasic heptahydrate</td>
 <td>VWR</td>
 <td>EMD-SX0715-1</td>
 <td>5g/L in water for making 50mM of buffer NaPi</td>
 </tr>
 <tr>	
 	<td>Magnesium sulfate heptahydrate</td>
 <td>VWR</td>
 <td>EM-MX0070-1</td>
 <td>2.5 g/L in water for making 1 L of infiltration media</td>
 </tr>
 <tr>	
 	<td>N-Lauroylsarcosine</td>
 <td>VWR</td>
 <td>TCL0151-500G</td>
 <td>0.1% v/v in GUS buffer</td>
 </tr>
 <tr>	
 	<td>5-Bromo-4-chloro-3-indoxyl-beta-D-glucuronide cyclohexylammonium salt (X-gluc)</td>
 <td>Gold Biotechnology</td>
 <td>G1281C</td>
 <td>1mg/100uL in methanol 100%</td>
 </tr>
 <tr>	
 	<td>4-Methylumbelliferyl-&mu;-D-glucuronide hydrate (MUG)</td>
 <td>Sigma</td>
 <td>M5664</td>
 <td>2 mM in 100 uL of GUS extraction buffer</td>
 </tr>
 <tr>	
 	<td>Potassium ferrocyanide trihydrate</td>
 <td>VWR</td>
 <td>EM-PX1460-1</td>
 <td>100mM stock for X-gluc substrate solution</td>
 </tr>
 <tr>	
 	<td>&beta;-Methylumbelliferone
(MU)
</td>
 <td>Sigma-Aldrich</td>
 <td>M1381</td>
 <td>For MU standard curve use GUS extraction buffer</td>
 </tr>
 <tr>	
 	<td>Sodium carbonate</td>
 <td>VWR</td>
 <td>EM-SX0395-11</td>
 <td>0.2 M in water for making GUS stop buffer</td>
 </tr>
 
 </tbody>
</table>

a &#946;-glucuronidase (gus) based cell death assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative positive or negative regulators of cell death. In this system, N. benthamiana leaves are co-infiltrated with a 35S driven expression cassette containing the gene to be analyzed, and the GUS vector pCAMBIA 2301 using Agrobacterium strain LBA4404 as a vehicle. Because live cells are required for GUS expression to occur, loss of GUS activity is expected when this marker gene is co-expressed with positive regulators of cell death. Equally, increased GUS activity is observed when anti-apoptotic genes are used compared to the vector control. As shown below, we have successfully used this system in our lab to analyze both pro- and anti-death players. These include the plant anti-apoptotic Bcl-2 Associated athanoGene (BAG) family, as well as, known mammalian inducers of cell death, such as BAX. Additionally, we have used this system to analyze the death function of specific truncations within proteins, which could provide clues on the possible post-translational modification/activation of these proteins. Here, we present a rapid and sensitive plant based method, as an initial step in investigating the death function of specific genes.

Watch this Scientific Journal Video about A Î²-glucuronidase GUS Based Cell Death Assay at JoVE.com

A &#946;-glucuronidase GUS Based Cell Death Assay

Programmed cell death assays commonly used in mammalian systems such as DNA laddering or TUNEL assays, are often difficult to reproduce in plants. In combination with a GUS reporter system, we propose a rapid, plant based transient assay to analyze the potential death properties of specific genes.

A &#946;-glucuronidase (GUS) Based Cell Death Assay

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, &beta;-glucuronidase (GUS), with putative ...

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