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 rise in the cytosol due to an influx of Ca²⁺ from extracellular and/or intracellular sources and generate a stimuli specific calcium signature^7,8,9. Ca²⁺ elevations in the cytosol are activated by many stimuli, but specificity is maintained by distinct stores releasing Ca²⁺, a unique Ca²⁺ signature and appropriate sensor proteins^10,11.

The use of alkylating agent, ethyl-methane sulfonate (EMS) for mutagenesis is a powerful tool in classical forward genetic screens to identify multiple independent genes involved in a process. EMS is a chemical mutagen predominantly inducing C to T and G to A transitions randomly throughout the genome and produces a 1 bp change in every 125 kb of the genome. EMS mutagenesis will induce ≈1000 single base pair changes, either insertion/deletions (InDel) or single nucleotide polymorphism (SNP) per genome¹². EMS-induced mutations are multiple point mutations with a mutation frequency ranging from 1/300 to 1/30000 per locus. This reduces the number of M₁ plants needed to find a mutation in a given gene. A M₁ seed population range of 2000-3000 is typically used to obtain mutations of interest in Arabidopsis thaliana^13,14.

Aequorin transgenics are Arabidopsis Columbia-0 (Col-0) ecotype plants expressing p35S-apoaequorin (pMAQ2) in the cytosol¹⁵. Aequorin is a Ca²⁺ binding protein composed of apoprotein and a prosthetic group consisting of luciferin molecule, coelenterazine. The binding of Ca²⁺ to aequorin, which has three Ca²⁺ binding EF-hands sites, results in coelenterazine being oxidized and cyclized to give the dioxetanone intermediate, followed by a conformational change of the protein accompanied by the release of carbon dioxide and singlet-excited coelenteramide¹⁶. The coelenteramide so produced emits a blue light (λ_max, 470 nm) that can be detected by the luminometer¹⁷. The extremely fast Ca²⁺ elevations can thus be measured in real time, and exploited for rapid forward genetic screens. This protocol aims to use the specificity of calcium response to identify novel key players that are involved in the Ca²⁺ signature. To achieve this task, we use EMS mutagenesis in transgenic aequorin and identify the SNPs associated with altered Ca²⁺ signaling. The protocol identifies mutants that show no or reduced Ca²⁺ elevations upon stimuli addition. These mutants can then be mapped to identify the genes responsible for the Ca²⁺ response. The method is applicable to any kind of liquid stimuli in plants that results in a Ca²⁺ elevation. Since Ca²⁺ elevation is one of the first responses in the plant defense signaling pathway, the identification of upstream response components can provide candidates for genetic engineering to develop resilient plants.

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 a waste container containing 1 M NaOH in equal volumes (NaOH helps to neutralize/inactivate EMS making it safe to discard). Discard the used plasticware in a 1 M NaOH solution.
   NOTE: After 24 h, dispose the discards according to hazardous material laboratory practices.
5. Wash the mutagenized seeds thoroughly with 40 mL of autoclaved water at least 8 times. Discard EMS containing water in a NaOH waste container as mentioned above.
6. For the final wash, add 40 mL of 100 mM sodium thiosulfate and rinse 3 times to remove traces of EMS.
7. Soak the seeds in 40 mL of autoclaved water for ~1 h to diffuse EMS out of seeds and then place them on filter paper until completely dry.
8. Transfer the seeds to a microcentrifuge tube and stratify the seeds at 4 °C in 40 mL of autoclaved water for 2-4 days. This helps in breaking seed dormancy and ensures homogenous growth.
9. Transfer both the mutagenized seeds (M₀) and the control seeds on to soil (soil composition: agropet: soilrite, 1:1) and transfer them to growth rooms with a 16 h light/8 h dark photoperiod, a light intensity of 150 μmol∙m⁻²∙s⁻¹ and ~70% relative humidity.
10. To determine if mutagenesis was successful, look for reduced germination speed and seedling growth, and chlorophyll sectoring¹⁸ (Figure 1A). Compare the mutagenized plants to the control plants to identify these physiological and developmental differences.
    NOTE: Different methods can be used for harvesting seeds from M₁ plants. In this protocol, we have used the single pedigree-based seed collection method. Each M₁ plant is given a unique number, starting from A1 to A3500.
11. Maintain individually numbered plants as discrete plant lines (Figure 1B).
12. Upon maturation, harvest seeds from these individual mutant plants and store as individual M₁ lines (Figure 2). From the single pedigree-based seed collection, we obtained around 5000 M₁ lines out of which 3500 M₁ lines were screened.

2. High-throughput screening to select mutants (8 months)

1. Identify novel mutants based on the Ca²⁺ response to a selected stimulus. Here, we used H₂O₂ as an example.
2. For identifying mutants, screen the M₂ generation. Since recessive mutants segregate at 1/8 frequency in M₂ generation upon EMS mutagenesis¹⁴, screening of 8-12 M₂-segregating plants covers one M₁ line and identifies a homozygous recessive mutant (using 12 seedlings increases the probability of finding a mutant). From each independent M₁ line, test 12 M₂ seedlings for Ca²⁺ response to H₂O₂ (12 M₂ seedlings per M₁ line).
3. Use a high throughput seed sterilization and hydroponic plant growth protocol¹⁹. Place nearly 12-15 M₂ seeds per M₁ line in individual wells of a 24-well tissue culture plate and sterilize using chlorine gas (40 mL of 12% sodium hypochlorite and hydrochloric acid, 3:1, v/v) in a desiccator for 4 h in a fume hood. After the procedure, open the desiccator and leave overnight for chlorine gas to evaporate.
4. After sterilization, bring plates outside and add liquid 1/2 MS media (half-strength MS without agar) to individual wells. Seal the plates with parafilm and stratify the seeds for 2-4 days at 4 °C and then move seeds to a growth chamber with 10 h light/ 14 h dark photoperiod at 20-22 °C with 70% relative humidity.
5. Once the seedlings are 8-12 days old, place 12 M₂ seedling from each line individually in a 96-well luminometer plate. For measuring Ca²⁺ response to H₂O₂, use a luminometer plate reader.
6. After seedling transfer, add 150 µL of 5-10 µM coelentrazine solution (diluted in autoclaved water from a 5 mM stock in methanol) (CAUTION) in individual wells in a dark/low-light area and store in dark at 21 °C for 8 h.
   NOTE: Coelentrazine is a prosthetic group that binds to apo-aequorin and reconstitutes it to functional aequorin. Coelentrazine is light sensitive and is hence stored in dark colored bottles, protected from light.
7. The next day, perform mutant screen using 10 mM H₂O₂ as a stimulus and measure the subsequent Ca²⁺ response.
8. For simultaneous measurement of 24 wells, create an automated kinetic program that measures the background for 1 min, followed by stimuli addition (40 μL) and measurement for 10 min, followed by total aequorin discharge (2 M CaCl₂ in 150 µL 20% ethanol) for 1-2 min.
   NOTE: An end discharge for the total aequorin is needed to quantify the measured Ca²⁺ and as additional control for functional aequorin. The end discharge is a short run of 1-2 min and does not cause significant plant death. Alternatively, if the plants die after discharge step, then re-screen the specific M₁ line and rescue without discharge. Such mutants can be confirmed in M₃ and M₄ generations using the discharge step.
9. Use a 24-well format scanning method that measures each row in 7 s interval with 300 ms integration time per well per measurement point. Use a wild-type seedling as control in each row for comparison and evaluation of the mutant.
   NOTE: A single 96 well plate containing M₂ seedlings will cover 8 individual M₁ line (8 M₁*12= 96 M₂) and can be screened in 2.5 h and each day 32 M₁ lines would be screened, 640 M₁ plants per month. The whole screening procedure after the plants are ready would take around 8 months.
10. Identify mutants based on loss of or reduced Ca²⁺ response with H₂O₂. Rescue the selected mutants through an antibiotic-based washing process. Wash the seedling with 25 mg/L cefotaxime solution twice and then transfer to rescue medium that contains 25 mg/L cefotaxime in 1/2 MS agar.
11. Grow the plates at a 10 h light/ 14 h dark photoperiod to obtain a mutant plant. The cefotaxime wash helps to remove any harmful micro-organisms on the seedling. Since the seedlings after reconstitution are kept un-sealed, minimize contamination when transferring back to sterile condition.
12. Transfer the mutant plant to soil for obtaining the homozygous M₃ population.

3. Data analysis and mutant identification (1-3 months)

NOTE: The readings provided from the above method are in relative light units (RLU).

1. For calculating [Ca²⁺]_cyt concentration, use the following concentration equation²⁰.
   pCa = 0.332588(−log (L/L_max)) + 5.5593
   Luminescence counts () and total remaining counts (_max).
2. Convert the obtained pCa values to [Ca²⁺]_cyt values in μM by multiplying by 10⁶. Then graphically plot against an aequorin (transgenic Col-0 aequorin) control for identifying putative H₂O₂ mutants to Ca²⁺ response.
3. Rescue seedlings showing a loss of or reduced response to H₂O₂ application rescued.
4. Upon maturation, harvest seeds from the rescued mutant and re-screen for response to H₂O₂ in M₃ seedlings. If all 12 M₃ seedlings show a Ca²⁺ response similar to the mother plant, consider the population to be a homozygous M₃ mutant population. If all 12 seedlings do not show such a response, then take them to M₄ generation and re-screen to obtain homozygous population.
5. Once a homozygous plant line has been obtained, identify the gene leading to altered phenotype using next generation sequencing.

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