A β-glucuronidase (GUS) Based Cell Death Assay

Summary

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.

Abstract

We have developed a novel transient plant expression system that simultaneously expresses the reporter gene, β-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.

Protocol

Nicotiana benthamiana plants are grown in a temperature-controlled growth chamber at 25°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

1. Streak LB/Rifampicin (25 μg/ml)/Kanamycin (100 μg/ml) agar plates with glycerol stocks of Agrobacterium tumefaciens (strain LBA4404) containing the appropriate vectors with the gene (s) to be assayed for cell death and the vector containing the GUS cassette under a constitutive promoter. Always inc....

Discussion

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.......

Materials

List of materials used in this article

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