Name: Video: Measuring Immune-Induced Inhibition of Seedling Growth - Concept
Uploaded: 2024-01-31T13:35:57.000+00:00
Duration: 1 min 30 s
Description: 64 Views. Source: Bredow, M. et al., Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana. J. Vis. Exp. (147),(2019). This video demonstrates the seedling growth inhibition assay in the Arabidopsis thaliana plant. The impact of increased concentration of immune peptide elicitor on seedling growth is determined.

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1. Seedling Growth Inhibition Assay
<ol>
	<li>Sterilize seeds and sow on Murashige and Skoog (MS) plates.
	<ol>
		<li>Prepare sterile half-strength (0.5x) MS medium (2.16 g/L) containing 0.8% agar [w/v]. Pour media into plates (90 x 15 mm) in a laminar flow hood.</li>
		<li>Sterilize approximately 100 seeds in a microcentrifuge tube by washing with 1 mL of 70% ethanol for 2 min and remove by aspiration. Add 1 mL of 40% bleach and gently rock for 17 mins at room temperature. Remove bleach by aspiration and wash three times in 1 mL of sterile water for 5 min. Resuspend in 1 mL of sterile 0.1% agar. 
		NOTE: Alternatively, use a chlorine gas seed sterilization protocol24.</li>
		<li>Sow approximately 100 seeds per genotype on MS agar plates using a pipette and seal with micropore tape in a laminar flow hood. 
		NOTE: Avoid placing seeds in close proximity as this will make transplanting seedlings difficult later.</li>
		<li>Stratify seeds by placing plates at 4 &#176;C (no light) for 3-4 days to synchronize germination (Figure 1A).</li>
	</ol>
	</li>
	<li>Transplant seedlings into 48-well plates containing immune elicitor peptides.
	<ol>
		<li>After stratification, move plates to light under short-day conditions (22 &#176;C, 10 h light, 150 &#956;E/m2/s light intensity, and 65-70% relative humidity) for 3-4 days to allow germination.</li>
		<li>In a laminar flow hood, prepare elicitor peptide dilutions (0 nM, 1 nM, 10 nM, 100 nM, and 1,000 nM) in sterile 0.5x MS liquid media containing 1% sucrose [w/v], using 25 mL of MS for each 48-well plate. Prepare plates by pipetting 500 &#181;L of MS liquid medium or MS medium containing peptides per well. If available, use a repeat pipettor for plate preparation to expedite the process. 
		NOTE: For each genotype, grow seedlings in MS without elicitor to account for any inherent differences in seedling growth.</li>
		<li>Using sterile forceps carefully transplant one seedling to each well of the same size and age, ensuring that there is no damage to the seedling or breakage to the root and that the root is submerged in media. For each genotype, transplant a minimum of 6-8 seedlings into each peptide dilution (Figure 1B). 
		NOTE: Transplant seedlings at 4 days post-germination, as short roots are easier to manipulate, and older seedlings may yield less optimal results.</li>
		<li>Seal plates with micropore tape and move back to light under standard short-day conditions (22 &#176;C, 10 h light, 150 &#956;E/m2/s light intensity, and 65-70% relative humidity). Allow seedlings to grow for 8-12 days.</li>
	</ol>
	</li>
</ol>
2. Determine percent growth inhibition.
<ol>
	<li>Carefully remove seedlings from 48-well plates and dry them by dabbing them on a paper towel. Weigh seedlings on an analytical scale and record values. If available, use an analytical scale equipped with USB output to record fresh weight values on a spreadsheet. Before weighing seedlings, take a photo to visually display growth inhibition (Figure 1C).</li>
	<li>Determine percent growth inhibition of elicitor-treated seedlings compared to seedlings grown in MS only as follows: 
	<img alt="Equation 1" src="/files/ftp_upload/21910/21910_Equation1.jpg" style="margin: auto;" /></li>
	<li>Plot data using a bar graph with standard error bars or using a box and whisker plot to better display inter-experimental variance.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>8-Well Sterile Plates with Lid</td>
			<td>Sigma-Aldrich</td>
			<td>CLS3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical Scale with Draft Sheid</td>
			<td>VWR</td>
			<td>VWR-225AC</td>
			<td>Any standard analytical scale can be used for growth inibition assays, however, a direct computer output is optimal.</td>
		</tr>
		<tr>
			<td>BioHit mLine Mechanical 12 Multichannel Pipette (30-300 uL</td>
			<td>Sartorius</td>
			<td>725240</td>
			<td>Any multichannel pipette can be used, as can a single pipetter if necessary.</td>
		</tr>
		<tr>
			<td>elf18 (AcSKEKFERTKPHVNVGTIG)</td>
			<td>EZ Biolab</td>
			<td>cp7211</td>
			<td>Store 10 mM stock peptide at -80 deg C in low protein binding tubes. When thawed, store 100 uM working stock at -20 deg C.</td>
		</tr>
		<tr>
			<td>Murisage and Skoog Basal Salts</td>
			<td>Cedarlane Labs</td>
			<td>MSP09-100LT</td>
			<td>Store at 4 deg C.</td>
		</tr>
		<tr>
			<td>White Polystyrene 96-Well Plates</td>
			<td>Fisher Scientific</td>
			<td>07-200-589</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring immune-induced inhibition of seedling growth

Source: Bredow, M. et al., <a href="https://app.jove.com/v/59437">Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana.</a> J. Vis. Exp. (147),(2019).
This video demonstrates the seedling growth inhibition assay in the Arabidopsis thaliana plant. The impact of increased concentration of immune peptide elicitor on seedling growth is determined.

<img alt="Figure 1" src="/files/ftp_upload/21910/21910_Figure1.jpg" /> 
Figure 1. Elicitor-induced seedling growth inhibition assay in Arabidopsis. (A) Sow seedlings on MS agar and grow for 3-4 days under standard short-day conditions. (B) Transplant seedlings to 48-well plates containing MS medium or MS containing different concentrations of elf18. (C) After 8-12 days, visually assess the seedling size and then measure fresh weight using an analytical scale...

Measuring Immune-Induced Inhibition of Seedling Growth

1. Seedling Growth Inhibition Assay

	Sterilize seeds and sow on Murashige and Skoog (MS) plates.
	
		Prepare sterile half-strength (0.5x) MS medium (2.16 ...

Transplant seedlings into wells containing desired concentrations of an immune elicitor peptide &#8212; a protein fragment derived from pathogen-associated molecules.
This peptide binds to the pattern recognition receptors, or PRRs, on the plant cells, initiating a signaling cascade.
This cascade includes phosphorylation of respiratory burst oxidase homolog, or RBOH proteins, that become enzymatically active and catalyze the production of reactive oxygen species or ROS.
ROS act as secondary messengers in immune signaling pathways, amplifying the defense response initiated by PRRs, and function as direct antimicrobial agents, inhibiting pathogen growth by damaging their cellular components.
During ROS production, the plant allocates energy and nutrients to support its immune response, diverting resources away from growth-related processes that result in growth inhibition.
Determine the percent growth inhibition in the elicitor-treated seedlings compared to untreated seedlings by correlating their fresh weights.
Increasing concentrations of peptide elicitors inhibit seedling growth due to heightened immune responses and associated biochemical alterations.

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64 Views. Source: Bredow, M. et al., Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana. J. Vis. Exp. (147),(2019). This video demonstrates the seedling growth inhibition assay in the Arabidopsis thaliana plant. The impact of increased concentration of immune peptide elicitor on seedling growth is determined. 

Video: Measuring Immune-Induced Inhibition of Seedling Growth - Concept

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Measuring Immune-Induced Inhibition of Seedling Growth

Transcript

Measuring Immune-Induced Inhibition of Seedling Growth

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay