Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Summary

A forward genetic screen based on Ca²⁺ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

Abstract

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca²⁺) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca²⁺ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M₁) and screening for the phenotype of interest was carried out in the segregating (M₂) population. Using a 96-well high-throughput Ca²⁺ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca²⁺ reporter background and identify novel Ca²⁺ regulated targets.

Introduction

A change in cytosolic calcium (Ca²⁺) concentration upon perception of biotic or abiotic stimulus is a well-studied early signaling event that activates many signaling pathways^1,2,3,4. A cell in its basal resting state maintains a lower Ca²⁺ concentration in the cytosol and sequesters excess Ca²⁺ in various intracellular organelles and extracellular apoplast leading to a steep Ca²⁺ gradient^5,6. Upon signal perception, Ca²⁺ levels ....

Protocol

1. EMS mutagenesis and single pedigree-based seed collection (1-3 months)

1. Weigh 150 mg of seeds (~7500) of aequorin for EMS mutagenesis (M₀ seeds). Weigh another 150 mg of seeds to be used as a control.
2. Transfer the seeds to a 50 mL tube and add 25 mL of 0.2% EMS (v/v) (CAUTION) (for treatment) or 25 mL of autoclaved water (for control).
   NOTE: Ethyl-methane sulfonate is a chemical agent for mutagenizing plant material.
3. Seal the tube with parafilm and wrap it in aluminum foil. Rotate the tube end-over-end for 18 h at room temperature.
4. Allow the seeds to settle. Remove the EMS solution carefully and discard in ....

Results

The EMS population was screened for H₂O₂ induced Ca²⁺ elevation. As discussed earlier, 12 individual M₂ seedlings were screened from each M₁ line. In Figure 3, one such M₁ line is plotted with each panel showing 12 individual M₂ seedlings. A wild-type aequorin is used as control for comparing and evaluating the mutant response. A recessive mutant segregates in the ratio of 1:7 (mutant: non mutant). When screening 12.......

Discussion

EMS mutagenesis is a powerful tool to generate mutations in population. The classical forward genetic screens using EMS has been an effective tool to identify novel genes for two major reasons: firstly, they do not require any prior assumptions on gene identity and secondly, they do not introduce any bias. There are several methods to generate a screening populations like EMS, T-DNA insertions, radiations etc. Out of all the methods, EMS-based mutagenesis has few advantages over the other methods. First, it is easier to .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
24 well tissue culture plate	Jetbiofil	11024	for growing seedlings
96 well white cliniplate	Thermo Scientific	9502887	for luminometer measurements
Aequorin
Agropet	Lab Chem India		for plant growth
Calcium chloride	Fisher Scientific	12135	for discharge solution
Coelenterazine	PJK	55779-48-1	prosthetic group for aequorin
Ehtylmethane sulfonate	Sigma Aldrich	M0880-5G	for seed mutagenesis
Ethanol	Analytical reagent	1170	for discharge solution
Hydrochloric acid	Merck Life Sciences	1.93001.0521	sterlization solution
Hydrogen peroxide	Fisher Scientific	15465	as stimulus for Calcium elevation
Luminoskan ascent	Thermo Scientific	5300172	aequorin luminescence measurement
MES buffer	Himedia	RM1128-100G	plant growth
Murashige and skoog media	Himedia	PT021-25L	plant growth
Sodium hydroxide	Fisher Scientific	27805	for neutralizing EMS
Sodium hypochlorite	Merck Life Sciences	1.93607.5021	sterlization solution
Sodium thiosulfate	Fisher Scientific	28005	for seed washing in step 1.6
soilrite	Lab Chem India		for plant growth
Square pots	Lab Chem India		for plant growth
Sucrose	Sigma Aldrich	S0389	plant growth
Taxim	Alkem	7180720	for seedling rescue