Name: Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling (Video) | JoVE Journal
Uploaded: 2020-08-01T13:00:00.000+00:00
Duration: 8 min 46 s
Description: Discover forward genetic screening using EMS mutagenesis and aequorin calcium reporters in Arabidopsis. This method identifies novel genes involved in plant calcium signaling and defense responses.

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5 minutes

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).

1. EMS mutagenesis and single pedigree-based seed collection (1-3 months)
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Seal the tube with parafilm and wrap it in aluminum foil. Rotate the tube end-over-end for 18 h at room temperature.</li>
	<li>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:&#160;After 24 h, dispose the discards according to hazardous material laboratory practices.</li>
	<li>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.</li>
	<li>For the final wash, add 40 mL of 100 mM sodium thiosulfate and rinse 3 times to remove traces of EMS.</li>
	<li>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.</li>
	<li>Transfer the seeds to a microcentrifuge tube and stratify the seeds at 4 &#176;C in 40 mL of autoclaved water for 2-4 days. This helps in breaking seed dormancy and ensures homogenous growth.</li>
	<li>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 &#956;mol&#8729;m&#8722;2&#8729;s&#8722;1 and ~70% relative humidity.</li>
	<li>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.</li>
	<li>Maintain individually numbered plants as discrete plant lines (Figure 1B).</li>
	<li>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.</li>
</ol>
2. High-throughput screening to select mutants (8 months)
<ol>
	<li>Identify novel mutants based on the Ca2+ response to a selected stimulus. Here, we used H2O2 as an example.</li>
	<li>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).</li>
	<li>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.</li>
	<li>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 &#176;C and then move seeds to a growth chamber with 10 h light/ 14 h dark photoperiod at 20-22 &#176;C with 70% relative humidity.</li>
	<li>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.</li>
	<li>After seedling transfer, add 150 &#181;L of 5-10 &#181;M 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 &#176;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.</li>
	<li>The next day, perform mutant screen using 10 mM H2O2 as a stimulus and measure the subsequent Ca2+ response.</li>
	<li>For simultaneous measurement of 24 wells, create an automated kinetic program that measures the background for 1 min, followed by stimuli addition (40 &#956;L) and measurement for 10 min, followed by total aequorin discharge (2 M CaCl2 in 150 &#181;L 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Transfer the mutant plant to soil for obtaining the homozygous M3 population.</li>
</ol>
3. Data analysis and mutant identification (1-3 months)
NOTE: The readings provided from the above method are in relative light units (RLU).
<ol>
	<li>For calculating [Ca2+]cyt concentration, use the following concentration equation20. 
	pCa = 0.332588(&#8722;log (L/Lmax)) + 5.5593 
	Luminescence counts () and total remaining counts (max).</li>
	<li>Convert the obtained pCa values to [Ca2+]cyt values in &#956;M by multiplying by 106. Then graphically plot against an aequorin (transgenic Col-0 aequorin) control for identifying putative H2O2 mutants to Ca2+ response.</li>
	<li>Rescue seedlings showing a loss of or reduced response to H2O2 application rescued.</li>
	<li>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.</li>
	<li>Once a homozygous plant line has been obtained, identify the gene leading to altered phenotype using next generation sequencing.</li>
</ol>

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</ol>

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

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 ...

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 &#8776;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.&#160;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 (&#955;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.

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

forward genetic screen using transgenic calcium reporter aequorin to identify novel targets in calcium signaling

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

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.

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...

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.

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...

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Research

JoVE Journal

Biology

This video introduces the concepts behind calcium imaging and includes an example protocol for time-lapse imaging of worms contained in molded agarose wells.

Calcium Imaging: A Method to Visualize Neural Activity in Live C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

In vivo Neuronal Calcium Imaging in C. elegans

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Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

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In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

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Ratiometric Calcium Imaging of Individual Neurons in Behaving Caenorhabditis Elegans

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Manipulation of Gene Function in Mexican Cavefish

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Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We describe here, the establishment and application of an Tg(Myh6-cre)1Jmk/J /Gt(ROSA)26Sortm38(CAG-GCaMP3)Hze/J (referred to as αMHC-Cre/Rosa26A-Flox-Stop-Flox-GCaMP3 below) mouse reporter line for cardiac reprogramming assessment. Neonatal cardiac fibroblasts (NCFs) isolated from the mouse strain are converted into induced cardiomyocytes (iCMs), allowing for convenient and efficient evaluation of reprogramming efficiency and functional maturation of iCMs via calcium (Ca2+) flux.

In vitro Assessment of Cardiac Reprogramming by Measuring Cardiac Specific Calcium Flux with a GCaMP3 Reporter

The present protocol describes all-optical control and observation of triggered cellular activity in iPSC-derived cardiomyocytes (iPSC-CMs) for high throughput drug screening and toxicity testing. Multi-parametric quantification of phenotypic patterns in time, and space, are shown. Long-term effects of drugs over hours, or sequential measurements over days, are demonstrated.

High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening

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A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

Retinal prostheses have the ability to generate visual perceptions. To advance the development of new prostheses, ex vivo methods are needed to test devices before implantation. This article provides a comprehensive protocol for studying calcium activity in the retinal ganglion cell layer when subjected to electrical stimulation.

Calcium Imaging In Electrically Stimulated Flat-Mounted Retinas

Discover forward genetic screening using EMS mutagenesis and aequorin calcium reporters in Arabidopsis. This method identifies novel genes involved in plant calcium signaling and defense responses.

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

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