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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.

measuring-immune-induced-inhibition-of-seedling-growth

Research

JoVE Encyclopedia of Experiments

Immunology

Immunodiagnostics

In Vitro and In Vivo Models

64 Views. المصدر: Bredow، M. et al. ، فحوصات تثبيط نمو الشتلات وانفجار الأكسدة التي يسببها النمط في نبات الأرابيدوبسيس ثاليانا. J. Vis. Exp. (147) ، (2019). يوضح هذا الفيديو مقايسة تثبيط نمو الشتلات في نبات نبات ن يتم تحديد تأثير زيادة تركيز محفز الببتيد المناعي على نمو الشتلات. 

Video: قياس تثبيط نمو الشتلات الناجم عن المناعة - Concept

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

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

تكلفة انخفاض مرونة نظام الزراعة المائية لتقييم استجابات النبات للجزيئات الصغيرة في ظروف معقمة

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

JoVE Journal

Environment

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

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend themselves against pathogen attack. The contribution of a specific Arabidopsis gene to the overall plant immune response can be specifically and quantitatively assessed by assaying the pathogen growth within the infected tissue. For over three decades, the hemibiotrophic bacterium Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326) has been widely applied as the model pathogen to investigate the molecular mechanisms underlying the Arabidopsis immune response. To deliver pathogens into the leaf tissue, multiple inoculation methods have been established, e.g., syringe infiltration, dip inoculation, spray, vacuum infiltration, and flood inoculation. The following protocol describes an optimized syringe infiltration method to deliver virulent Psm ES4326 into leaves of adult soil-grown Arabidopsis plants and accurately screen for enhanced disease susceptibility (EDS) towards this pathogen. In addition, this protocol can be supplemented with multiple pre-treatments to further dissect specific immune defects within different layers of plant defense, including Salicylic Acid (SA)-Triggered Immunity (STI) and MAMP-Triggered Immunity (MTI).

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

Quantification of pathogen growth is a powerful tool to characterize various Arabidopsis thaliana (hereafter: Arabidopsis) immune responses. The method described here presents an optimized syringe infiltration assay to quantify the Pseudomonas syringae pv. maculicola ES4326 growth in adult Arabidopsis leaves.

بكتيريا ورقة تسلل الفحص لتوصيف غرامة الردود الدفاع النبات باستخدام<em&gt; نبات الأرابيدوبسيس thaliana-الزائفة syringae</em&gt; Pathosystem

In the absence of specialized mobile immune cells, plants utilize their localized programmed cell death and Systemic Acquired Resistance to defend ...

Immunology and Infection

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

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. The tomato-P. syringae pv. tomato pathosystem is widely used to dissect the genetic basis of plant innate responses and disease resistance. While disease was successfully managed for many decades through the introduction of the Pto/Prf gene cluster from Solanum pimpinellifolium into cultivated tomato, race 1 strains of P. syringae have evolved to overcome resistance conferred by the Pto/Prf gene cluster and occur worldwide.
Wild tomato species are important reservoirs of natural diversity in pathogen recognition, because they evolved in diverse environments with different pathogen pressures. In typical screens for disease resistance in wild tomato, adult plants are used, which can limit the number of plants that can be screened due to their extended growth time and greater growth space requirements. We developed a method to screen 10-day-old tomato seedlings for resistance, which minimizes plant growth time and growth chamber space, allows a rapid turnover of plants, and allows large sample sizes to be tested. Seedling outcomes of survival or death can be treated as discrete phenotypes or on a resistance scale defined by amount of new growth in surviving seedlings after flooding. This method has been optimized to screen 10-day-old tomato seedlings for resistance to two P. syringae strains and can easily be adapted to other P. syringae strains.

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

The seedling flood assay facilitates rapid screening of wild tomato accessions for the resistance to the Pseudomonas syringae bacterium. This assay, used in conjunction with the seedling bacterial growth assay, can assist in further characterizing the underlying resistance to the bacterium, and can be used to screen mapping populations to determine the genetic basis of resistance.

تعريف عالي الإنتاجية للمقاومة لـ Pseudomonas syringae pv. الطماطم في الطماطم باستخدام الشتلات الفيضانات أساي

Tomato is an agronomically important crop that can be infected by Pseudomonas syringae, a Gram-negative bacterium, resulting in bacterial speck disease. ...

Measuring Immune-Induced Inhibition of Seedling Growth

Transcript

Measuring Immune-Induced Inhibition of Seedling Growth