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

Deepika Mittal; Shruti Mishra; Ramgopal Prajapati; Jyothilakshmi Vadassery

doi:10.3791/61259

Biology

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

Published: August 1, 2020 doi: 10.3791/61259

Deepika Mittal¹, Shruti Mishra¹, Ramgopal Prajapati¹, Jyothilakshmi Vadassery¹

¹National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India

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 pathways¹^,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⁺ gradient⁵^,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 signature⁷^,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 proteins¹⁰^,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 thaliana¹³^,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.

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Protocol

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

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.
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.
Seal the tube with parafilm and wrap it in aluminum foil. Rotate the tube end-over-end for 18 h at room temperature.
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.
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.
For the final wash, add 40 mL of 100 mM sodium thiosulfate and rinse 3 times to remove traces of EMS.
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.
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.
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⁻¹and ~70% relative humidity.
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.
Maintain individually numbered plants as discrete plant lines (Figure 1B).
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)

Identify novel mutants based on the Ca²⁺response to a selected stimulus. Here, we used H₂O₂as an example.
For identifying mutants, screen the M₂generation. 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).
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.
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.
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.
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.
The next day, perform mutant screen using 10 mM H₂O₂ as a stimulus and measure the subsequent Ca²⁺response.
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.
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.
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.
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.
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).

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).
Convert the obtained pCa values to [Ca²⁺]_cytvalues 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.
Rescue seedlings showing a loss of or reduced response to H₂O₂ application rescued.
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.
Once a homozygous plant line has been obtained, identify the gene leading to altered phenotype using next generation sequencing.

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Representative Results

The EMS population was screened for H₂O₂induced 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 individual seedlings per M₁ line, we can identify 1 or 2 mutants per line. We have identified 2 putative mutants from 12 M₂ seedlings (Figure 3). These mutants are further taken to M₃ and M₄ and a homozygous population is generated. The homozygous mutant is further mapped to identify the causal gene.

Figure 1: EMS mutagenized Arabidopsis. (A) To determine a successful EMS mutagenesis, we looked for chlorophyll sectoring in the mutagenized plant population (indicated by arrow). Statistically, 0.1 to 1% of M₁ plants must show chlorotic sectors. (B) Individual potting of M₁ plants was done to perform a single pedigree-based seed collection. Please click here to view a larger version of this figure.

Figure 2: A schematic representation of the forward genetic screen methodology. 7500 transgenic Arabidopsis plants expressing cytosolic Aequorin (Aeq) in Col-0 are mutagenized with ethyl methanesulphonate (EMS) in the M₀ generation. Around 5000 seedlings from M₁ generation are propagated individually by single pedigree method and propagated to M₂ generation. 12 plants from each M₂ line are screened for the phenotype of interest (around 3000-3500 M₁ lines). Mutant for the phenotype of interest is rescued and propagated to the M₃ generation and screened for homozygosity. Please click here to view a larger version of this figure.

Figure 3: Identification of mutants with altered response to H₂O₂ treatment. A representative figure to depict mutant selection from a single segregating M₁ line is shown. The panels show the screening result (12 segregating M₂ seedlings per individual M₁ line) upon stimulation with 10 mM H₂O₂. WT (Aequorin) control (green line), average of all 12 M₂ seedlings is the mean response (red line) and A1-X (black line) are individual seedlings numbered from 1-12. For each graph, the y-axis is the [Ca²⁺]_cyt (µM) and the x-axis is time (min). [Ca²⁺]_cyt levels were calculated from relative light units (RLUs). Please click here to view a larger version of this figure.

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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 generate a mutant population by exposure to EMS as described in the current protocol²¹^,²². Second, a sufficiently large number of mutant seed population can be generated, which can be used for multiple screens for one or more stimuli. Third, a weak allele of an essential gene generated due to a missense mutation can be identified. Fourth, an array of gene function effects can be identified using the EMS screen including complete loss-of-function, partial function loss, altered function and a constitutive gene function. It can help in identification of double mutants that is not feasible by other mutagenesis methods²³^,²⁴^,²⁵^.The single pedigree based M₁ seed collection used in this protocol is also advantageous as it allows one to go back to the mother population to identify the same mutant again, if the progeny is lost in the subsequent future generations. It offers the possibility for recovering mutations that are infertile when homozygous and can be recovered via the heterozygous siblings of the mutant plants. Secondly, this strategy guarantees the independence of all mutants isolated when compared to bulking of seeds in M₁ generation. It ensures that mutations isolated from the M₂ collection are different alleles at the same locus rather than the same mutational event²⁶.

The genes identified through EMS mutagenesis screens are dependent on the phenotype used for screening the population. The faster the phenotype of interest can be screened, the easier is to identify novel pathways. Ca²⁺ is a ubiquitous secondary messenger that is among the first signaling cascades to be activated. It acts as a mediator for plant response against a wide array of biotic and abiotic stimuli. Additionally, the calcium reporter aequorin can be localized to various sub-cellular compartments and organelles²⁷^,²⁸^,²⁹. This opens avenues for identifying roles of protein localized in these compartments in calcium response dynamics³⁰^,³¹^,³².

Forward genetic screens based on EMS-mutagenesis in aequorin and using Ca²⁺as readouts have remained contemporary since their discovery. The advantages of this method have outweighed the pitfalls. However, few limitations of the technique still need to be carefully evaluated. The screen is labor and time-intensive and requires identification of mutant plants from a vast mutagenized population. Hence, detailed planning based on resource availability, work personnel requirement and space constrains must be done before embarking on the experimental plan. The second major challenge with the aequorin-based Ca²⁺ screens is possibility of false positives without a discharge step. Hence a very short discharge step is included in the protocol. Random mutations can also lead to generation of sterile plants that cannot be rescued due to multiple mutations. Thirdly, Ca²⁺ signature is highly tissue specific and consistency in screening must be ensured⁴.

Not many forward genetic screens have identified receptors, channels, pumps and transporters of Ca²⁺ as use of Ca²⁺ as a screening phenotype in forward genetics was rare. Stimuli (e.g., H₂O₂) induced Ca²⁺elevation is used as marker for a forward genetic screen in our methodology, to identify new genetic components involved in the process. A similar strategy using EMS mutagenized aequorin population has led to the discovery of many receptors like DORN1 which is eATP receptor³³, calcium channel OSCA³⁴, LORE receptor involved in lipopolysaccharide sensing³⁵ and the ribonuclease PARN1³⁶. A recent study published by Wu et al. has used a very similar methodology of screening EMS-mutagenized aequorin plants upon H₂O₂ elicitation to identify the novel hydrogen peroxide sensor HPCA1³⁷. Hence the protocol using EMS mutagenesis in Ca²⁺reporter background is a promising method for novel gene discovery involved in stimuli sensing.

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Disclosures

None of the authors have any conflicts of interest to declare.

Acknowledgments

We thank National Institute of Plant Genome Research - Phytotron Facility for plant growth, Bombay Locale for the video shoot, and the Department of Biotechnology- eLibrary Consortium for providing access to e-resources. This work was supported by the Department of Biotechnology, India through the National Institute of Plant Genome Research Core Grant, Max Planck Gesellschaft-India Partner Group program; and CSIR-Junior Research Fellowship (to D.M and S.M) and Department of Biotechnology-Junior Research Fellowship (to R.P).

Materials

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

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