Work in our lab is funded through the Natural Resources and Engineering Research Council of Canada (NSERC) Discovery Program, the Canadian Foundation for Innovation John R. Evans Leader&#39;s Fund, and Queen&#39;s University. KS and IS are supported by tandem Ontario Graduate Scholarships and NSERC Canada Graduate Scholarships for master&#39;s students (CGS-M).

1. Detection of ROS burst in Arabidopsis leaf discs following immune elicitation
<ol>
	<li>Plant growth and maintenance.
	<ol>
		<li>To synchronize germination, stratify Arabidopsis seeds by suspending approximately 50 seeds in 1 mL of sterile 0.1% agar [w/v] and store at 4 &#176;C (no light) for 3-4 days. 
		NOTE:&#160;Stratify a wild type background control (for example, Col-0) and genotypes with high and low immune outputs (for example,&#160;cpk28-1&#160;and&#160;bak1-5,&#160;respectively) to serve as internal controls.</li>
		<li>Sow seeds on soil and germinate under standard short-day conditions (22 &#176;C, 10 h light, 150 &#956;E/m2/s light intensity, and 65-70% relative humidity).</li>
		<li>After approximately 7 days, use forceps to transplant individual seedlings to new pots, separated by at least 4 cm to permit full rosette development. Transplant 6-12 seedlings per genotype and return to standard short-day conditions. Regularly water and fertilize plants (1 g/L of 20-20-20 fertilizer every 2 weeks).</li>
	</ol>
	</li>
	<li>Collect leaf discs in 96-well plates for overnight recovery.
	<ol>
		<li>At 4-5 weeks post-germination (Figure 1A), use a sharp 4 mm biopsy punch to remove one leaf disc per plant, avoiding the mid-vein and being careful to limit wounding (Figure 1B). Place leaf discs in an unused 96-well luminometer plate containing 100 &#956;L of ddH2O with the adaxial side facing upwards to prevent desiccation22 (Figure 1C). If assessing multiple elicitors, remove 1 leaf disc from the same leaf for each elicitor treatment. 
		NOTE: Sample leaves that are fully expanded, third to fifth from the top of the rosette, and approximately the same size and age to limit variability. If possible, cut leaf discs in half using a razor blade prior to overnight recovery, as this increases the surface area exposed to elicitor solution23.</li>
		<li>Recover overnight at room temperature to prevent the interference of ROS produced by wounding during leaf disc collection. Cover plates with a lid to prevent evaporation.</li>
	</ol>
	</li>
	<li>Perform the elicitor treatment and measure ROS production.
	<ol>
		<li>Program the plate reader prior to adding the reaction solution, as this will reduce the time between ROS burst initiation and the first measurements recorded. Use a commercial software that is appropriate for the plate reader. In our case (see Table of Contents), click Settings, and select the LUM96 Cartridge and the Kinetic Read Type. Click the Read Area category and drag to select a subset of wells or the entire plate to be read. Under the PMT and Optics tab, set the integration time to 1,000 ms. Under the Timing tab, set the Total Run Time to 40-60 min and the Interval to 2 min.</li>
		<li>Remove water from each well using a multi-channel pipette. 
		NOTE: Do not puncture leaf discs, as this may cause wounding stress and result in more variable ROS outputs.</li>
		<li>Prepare a reaction solution containing 100 &#956;M luminol, 10 &#956;g/mL HRP, and the desired concentration of elicitor (for example: elf18 at 1 nM, 10 nM, 100 nM, or 1,000 nM) in sterile ddH2O using 10 mL of solution for one 96-well plate. 
		NOTE:&#160;Dissolve lyophilized peptides in sterile H2O in low-binding tubes to make a 10 mM stock, flash-freeze in liquid N2, and store at -80 &#176;C. When ready to use, dilute stocks in sterile H2O to generate a 100 &#956;M working stock and store at -20 &#176;C.</li>
		<li>Use a multi-channel pipette to dispense 100 &#956;L of the reaction mixture to each well, adding the solution to all leaf discs of the same treatment at the same time22. Include a control reaction (no elicitor) for each genotype to assess basal ROS levels in the absence of elicitation. Immediately measure light emission for all wavelengths in the visible spectrum using a microplate reader. 
		NOTE: Prepare and apply the reaction solution under low light, as luminol and HRP are light-sensitive reagents. Keep reagents on ice or at -20&#176;C unless in use.</li>
		<li>Measure light emission with a 1,000 ms integration time in 2 min intervals over a 40-60 min period in a microplate reader in order to capture the dynamic oxidative burst (Figure 1D). 
		NOTE: Use a longer integration time to improve assay sensitivity11, while ensuring that all samples in a 96-well plate can be measured within one interval.</li>
	</ol>
	</li>
	<li>Data interpretation.
	<ol>
		<li>For each genotype, use a spreadsheet application to calculate the average photon count and standard error at each time point, and display ROS production as a function of time using a scatter plot. 
		<img alt="Equation 1" src="/files/ftp_upload/59437/59437eq1.jpg" /> 
		Where t = each time point</li>
		<li>Alternatively, sum the photon counts for each leaf disc and present the average of these values for each genotype using a bar graph with standard error bars or a box and whisker plot. 
		<img alt="Equation 2" src="/files/ftp_upload/59437/59437eq2.jpg" /></li>
	</ol>
	</li>
</ol>
2. 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 2A).</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 2B). 
		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>
	<li>Determine percent growth inhibition.
	<ol>
		<li>Carefully remove seedlings from 48-well plates and dry by dabbing on 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 2C).</li>
		<li>Determine percent growth inhibition of elicitor-treated seedlings compared to seedlings grown in MS only (Figure 2D) as follows: 
		<img alt="Equation 3" src="/files/ftp_upload/59437/59437eq3.jpg" /></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>
	</li>
</ol>

This paper describes two methods for assaying pattern-triggered immune responses in Arabidopsis, offering quantitative approaches to evaluating immune output without the use of specialized equipment. In combination, pattern-triggered ROS and SGI can be used to assess early and late responses to microbe perception, respectively.
The major limitation of the oxidative burst assay is variability. For reasons that are not completely understood, absolute RLUs often differ by an order of magnitude between experiments. Because between-experiment variability is high, it is advisable to include internal reference controls with high (e.g., cpk28-1) and low (e.g., bak1-5) oxidative bursts in addition to a wild-type control (e.g., Col-0). However, measures can be taken to increase reproducibility between experiments. Environmental conditions such as temperature, humidity, photoperiod, and light intensity should be identical between replicates. The age and health of plants should also be considered when sampling. Leaf discs can be collected from plants that are between 4 and 7 weeks of age grown under short day conditions (6-10 hours of light). Anecdotally, the most consistent results were found with plants older than 6 weeks post germination but not yet flowering or senescing. It is important to grow plants in clean environmental chambers so that they are not exposed to common glasshouse pests such as powdery mildew or chewing insects. Since seedlings for SGI are grown in sterile MS media, and for a shorter time, environmental fluctuations are largely not a concern. However, variation can occur if seedlings become damaged while transplanting, if seedlings selected for elicitor treatments are different sizes, or if the growth media becomes contaminated. It is recommended to include the internal reference control genotypes described above in SGI experiments as well.
Elicitor concentration is another important consideration when conducting immune assays. Elicitors are perceived by PRRs resulting in a rapid and robust ROS burst peaking 10-20 minutes after elicitor treatment (Figure 2B). However, the magnitude of the burst is dependent on both the elicitor as well as the plant genotype. Therefore, an elicitor dose series, as presented in Figure&#160;3 and Figure&#160;4, is recommended in order to identify a suitable elicitor concentration. Pattern-triggered ROS can also be assayed by inoculating leaf discs with live microbes23,26 or microbial extracts26,27,28. For example, transient and dose-dependent ROS bursts can be detected in Arabidopsis in response to virulent pathovars of Pseudomonas syringae, peaking at 35-40 minutes and reaching basal levels approximately 70 mins after elicitation23,29. In response to avirulent bacteria29 or the fungal pathogen P. infestans30,31, a second more pronounced accumulation of ROS is produced that can be monitored 6-10 hours after inoculation. For weaker elicitors, such as fungal chitin32 or chitosan33, more sensitive luminescent indicators can be used, such as the luminol derivative L-012, however, the background signal produced is often higher32,34. Importantly, the plant ecotype will also dictate its responsiveness to specific elicitors35,36. For example, while bacterial flagellin is capable of eliciting immune responses in several Arabidopsis ecotypes including Col-0, the Wassilewskija (Ws-0) ecotype expresses a non-functional variant of the PRR FLAGELLIN-SENSING 2 (FLS2) and is therefore insensitive to flagellin14,37.
Immune-induced ROS can also be observed in leaf discs of other dicotyledonous plants including Brassica napus38, tomato39, Nicotiana benthamiana22,40,41, and several other Solanaceous species41. Additional luminol-based ROS detection assays have been developed for&#160;plants using tissue extracts10, cell suspension cultures8,42, and protoplasts43, and are particularly useful in systems where leaf disc protocols are not effective42. For example, elicitor-induced ROS bursts have been described using cell suspension cultures in rice42,44 and wheat45,46, as well as the gymnosperm Araucaria angustifolia47 and the moss Physcomitrella patens48. SGI has not been used as broadly to measure immune signaling. However, growth inhibition in response to elicitors has been demonstrated in N. benthamiana41 and B. napus38,49. Rapid growth inhibition has also been demonstrated in P. patens in response to fungal chitin occurring within 2 mins of exposure, which can be observed using time-lapse photography48.
With modification, both SGI and oxidative burst assays can be used for high-throughput screens to identify immune regulators. For example, mutagenized populations of Arabidopsis can be grown on MS medium and flooded with elicitors to identify insensitive mutants15,50,51,52,53. Alternatively, mutagenized populations can be assessed for elicitor-triggered ROS, which has been successfully executed with both leaf-discs54 and whole seedlings grown on MS plates19. Another useful screening method is the transient expression of proteins in N. benthamiana for ROS analysis prior to the development of transgenic overexpression lines22,40,55. However, intra-experimental variation is higher than in stable Arabidopsis lines due to differential protein expression in N. benthamiana leaves22, although this can be partially mitigated by infiltrating reference controls on the same leaf as experimental samples.
In summary, immune-induced oxidative burst and SGI assays are quick and reliable methods for assessing PRR-mediated signaling in Arabidopsis. These methods can be extended to other systems and used for large-scale screens to uncover novel immune regulators.

To perceive and defend against pathogens, plants have evolved membrane-bound pattern recognition receptors (PRRs) that detect conserved microbial molecules outside the cell known as microbe-associated molecular patterns (MAMPs)1. The binding of MAMPs to their cognate PRRs initiates protein kinase-mediated immune signaling resulting in broad-spectrum disease resistance2. One of the earliest responses following PRR activation is the phosphorylation and activation of integral plasma membrane RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) proteins that catalyze the production of extracellular reactive oxygen species (ROS)3,4. ROS play an important role in establishing disease resistance, acting both as secondary messengers to propagate immune signaling as well as direct antimicrobial agents5. The first observation of an immune-elicited oxidative burst was described using potato tubers of cv. Rishiri following Phytophthora infestans inoculation6. ROS production has been evaluated in several plant species using leaf discs7, cell suspension cultures8, and protoplasts6. Described here is a simple method for assaying pattern-triggered ROS production in leaf discs of Arabidopsis thaliana (Arabidopsis).
As a response to MAMP perception, activated RBOH proteins catalyze the production of superoxide radicals (O2-), hydroxyl radicals (&#8226;OH), and singlet oxygen (1O2) that are converted into hydrogen peroxide (H2O2) in the extracellular space9. H2O2 can be quantified by luminol-based chemiluminescence in the presence of the oxidizing agent horseradish peroxidase (HRP)10. HRP oxidizes H2O2 generating a hydroxide ion (OH&#8722;) and oxygen gas (O2) which react with luminol to produce an unstable intermediate that releases a photon of light10. Photon emission can be quantified as relative light units (RLUs) using a microplate reader or imager capable of detecting luminescence, which have&#160;become standard pieces of equipment in most molecular laboratories. By measuring the light produced over a 40-60-minute interval, a transient oxidative burst can be detected as early as 2-5 minutes after the elicitor treatment, peaking at 10-20 minutes, and returning to basal levels after ~60 minutes11. The cumulative light produced over this time course can be used as a measure of immune strength corresponding to the activation of RBOH proteins12. Conveniently, this assay does not require specialized equipment or cumbersome sample preparation.
Peaking shortly after MAMP detection, the oxidative burst is considered an early immune response, along with MAPK activation and ethylene production5. Later immune responses include transcriptional reprogramming, stomatal closure, and callose deposition2,5. Prolonged exposure to MAMPs continually activates energetically-costly immune signaling resulting in the inhibition of plant growth, indicative of a trade-off between development and immunity13. Pattern-triggered seedling growth inhibition (SGI) is widely used to assess immune output in Arabidopsis and has been integral to the identification of several key components of immune signaling including PRRs14,15,16. Therefore, this paper additionally presents an assay for&#160;pattern-triggered SGI in Arabidopsis, whereby seedlings are grown in multi-well plates containing standard media or media supplemented with an immune elicitor for 8-12 days and then weighed using an analytical scale.
To demonstrate how ROS and SGI assays can be used to monitor PRR-mediated signaling, three genotypes that represent varying immune outputs were chosen: (1) the wild type Arabidopsis accession Columbia (Col-0), (2) the dominant-negative bak1-5 mutant in which the multi-functional PRR co-receptor BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1&#160;(BAK1) is non-functional in immune signaling17,18, and (3) the recessive cpk28-1 mutant, which lacks the regulatory protein CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28) and displays heightened immune-triggered responses19,20. ROS and SGI assays are presented in response to a synthetically-produced elf18 peptide epitope of bacterial Elongation Factor Tu (EF-Tu), recognized in Arabidopsis by the PRR EF-Tu RECEPTOR (EFR)15. These protocols can be used with other immune elicitors such as the bacterial motility protein flagellin14 or endogenous Plant Elicitor Proteins (AtPeps)16, however, it should be noted that plant responsiveness differs depending on the elicitor21. Together, ROS and SGI assays can be used for the quick and quantitative assessment&#160;of early and late PRR-mediated responses.

<table><tbody><tr><td>20-20-20 Fertilizer</td><td>Plant Prod</td><td>10529</td><td>Mix 1g/L in water and apply to plants every 2 weeks for optimal growth.</td></tr><tr><td>4 mm Biopsy Punch</td><td>Medical Mart</td><td>232-33-34-P</td><td>A cork borer set with a 0.125 cm^2 surface area can also be used.</td></tr><tr><td>48-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 (Ac-SKEKFERTKPHVNVGTIG)</td><td>EZ Biolab</td><td>cp7211</td><td>Store 10 mM stock peptide at -80C in low protein binding tubes. When thawed, store 100 uM working stock at -20C.</td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>22-327379</td><td></td></tr><tr><td>Horseradish Peroxidase</td><td>Sigma-Aldrich</td><td>P6782</td><td>Dissolve in pure water. Store at -20C and away from light.</td></tr><tr><td>Luminol</td><td>Sigma-Aldrich</td><td>A8511</td><td>Dissolve in DMSO. Store at -20C and away from light.</td></tr><tr><td>Murisage and Skoog Basal Salts</td><td>Cedarlane Labs</td><td>MSP09-100LT</td><td>Store at 4C.</td></tr><tr><td>Soil</td><td>SunGrow Horticulture</td><td>Sunshine Mix #1</td><td>Other soil types can also be used to grow Arabidopsis. Mix with water when filling pots.</td></tr><tr><td>SpectraMax Paradigm Multi Mode Microplate Reader with LUM Module</td><td>Molecular Devices</td><td>Must request a quote</td><td>Any plate reader capable of detecting luminescence can be used for these assays.</td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389-1KG</td><td>Store at room temperature.</td></tr><tr><td>White Polystyrene 96-Well Plates</td><td>Fisher Scientific</td><td>07-200-589</td><td></td></tr></tbody></table>

pattern-triggered oxidative burst and seedling growth inhibition assays in arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Mutant cpk28-119,25 and bak1-517,18 plants were used to demonstrate expected outcomes for genotypes with high and low immune responses, respectively, in oxidative burst and SGI assays relative to a wild-type background control (Col-0). To assess dose-dependent effects, a 10-fold peptide dilution series (1-1,000 nM) of elf18 was used. As expected, cpk28-1 loss-of-function lines had a higher cumulative (Figure 3A) and average (Figure 3B) ROS burst compared to Col-0, whereas bak1-5 displayed reduced ROS production at concentrations between 10 nM and 1,000 nM (Figure 3). Expected differences in SGI could be discerned between all genotypes grown in 100 nM and 1,000 nM elf18 (Figure 4A), which could also be visually observed in the 1,000 nM elf18 treatment (Figure 4B). Characteristic of high immune signaling, cpk28-1 mutants were markedly smaller than Col-0 when grown in 1,000 nM elf18, while bak1-5 mutants displayed weak growth inhibition relative to Col-0 due to disrupted MAMP detection.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59437/59437fig1.jpg" /> 
Figure 1. Luminol-based oxidative burst assay following immune induction in Arabidopsis. (A) Grow plants on soil in short day conditions for 4-5 weeks. (B) Use a 4 mm biopsy punch to collect leaf discs from each plant and recover overnight in ddH2O. (C) Add reaction solution (100 &#956;M luminol, 10 &#956;g/mL HRP, and the desired concentration of elicitor) and measure light emission over 40-60 min, in 2 min intervals with an integration time of 1,000 ms. (D) Determine average photon counts for each genotype relative to Col-0 (shown in green), a high ROS control such as cpk28-1 (shown in purple), and a low ROS control such as bak1-5 (shown in orange). <a href="https://www.jove.com/files/ftp_upload/59437/59437fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59437/59437fig2.jpg" /> 
Figure 2. 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 to determine percent growth inhibition. <a href="https://www.jove.com/files/ftp_upload/59437/59437fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59437/59437fig3.jpg" /> 
Figure 3. Representative elf18-induced oxidative burst in three Arabidopsis genotypes. Four-week-old plants were treated with&#160;a dilution series of elf18 (0 nM &#39;mock&#39;, 1 nM, 10 nM, 100 nM, and 1,000 nM). Col-0 represents the wild-type background control, with cpk28-1 and bak1-5 representing high and low ROS phenotypes, respectively. (A) Total photon count represented as relative light units following elf18 treatment (n=6 leaf discs from independent plants). Statistical differences are represented by lower-case letters and were calculated using a one-way ANOVA with a post-hoc Tukey&#39;s Honest Significant Difference test (p&#60;0.05). (B) Average photon count, represented as relative light units, over 40 min following elf18 treatment (n=6 leaf discs from independent plants). Error bars represent standard error of the mean. Similar results were obtained in two of three experiments. <a href="https://www.jove.com/files/ftp_upload/59437/59437fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59437/59437fig4.jpg" /> 
Figure 4. Representative elf18-induced seedling growth inhibition in three Arabidopsis genotypes. Wild-type (Col-0), cpk28-1, and bak1-5 seedlings were grown in a 10-fold dilution series (0-1,000 nM) of elf18. (A) Percent growth inhibition was calculated by comparing the weight of individual seedlings grown in elf18 (n=6 individual seedlings) to the average weight of seedlings of the same genotype grown in MS only (&#39;mock&#39;) (n=6 individual seedlings) over a 10-day period. Statistical differences are represented by lower-case letters and were calculated using a one-way ANOVA with a post-hoc Tukey&#39;s Honest Significant Differences test (p&#60;0.05). (B) Visual demonstration of SGI in two representative seedlings of Col-0, cpk28-1, and bak1-5 in response to increasing concentrations of elf18. Similar results were obtained in three of four experiments. <a href="https://www.jove.com/files/ftp_upload/59437/59437fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana at JoVE.com

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

pattern-triggered-oxidative-burst-seedling-growth-inhibition-assays

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

13.1K Views.  Queen's University. This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

Video: Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize conserved microbial features called pathogen-associated molecular patterns (PAMPs) and this detection leads to PAMP-triggered immunity (PTI), which effectively prevents colonization of plant tissues by non-pathogens1,2. The most well studied system in PTI is the FLS2-dependent pathway3. FLS2 recognizes the PAMP flg22 that is a component of bacterial flagellin. 
Successful pathogens possess virulence factors or effectors that can suppress PTI and allow the pathogen to cause disease1. Some plants in turn possess resistance genes that detect effectors or their activity, which leads to effector-triggered immunity (ETI)2.
We describe a cell death-based assay for PTI modified from Oh and Collmer4. The assay was standardized in N. benthamiana, which is being used increasingly as a model system for the study of plant-pathogen interactions5. PTI is induced by infiltration of a non-pathogenic bacterial strain into leaves. Seven hours later, a bacterial strain that either causes disease or which activates ETI is infiltrated into an area overlapping the original infiltration zone. PTI induced by the first infiltration is able to delay or prevent the appearance of cell death due to the second challenge infiltration. Conversely, the appearance of cell death in the overlapping area of inoculation indicates a breakdown of PTI. 
Four different combinations of inducers of PTI and challenge inoculations were standardized (Table 1). The assay was tested on non-silenced N. benthamiana plants that served as the control and plants silenced for FLS2 that were predicted to be compromised in their ability to develop PTI.

Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants

A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

To perceive potential pathogens in their environment, plants use pattern recognition receptors (PRRs) present on their plasma membranes. PRRs recognize ...

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, phloem transport and vascular patterning. This protocol shows bottom-to-top carboxyfluorescein (CF) movement in the Arabidopsis by using the root-cutting and the hypocotyl-pinching procedure respectively. These two different procedures result in different efficiencies of CF movement: about 91% appearance of CF in the shoots with the hypocotyl-pinching procedure, whereas only about 70% appearance of CF with the root-cutting procedure. The simple change of loading sites, resulting in significant changes in the mobile efficiency of this symplastic dye, suggests CF movement might be subject to the symplastic regulation, most probably by the root-hypocotyl junction.

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

The goal of this protocol is to show how to load the CFDA into different sites of the bottom parts of Arabidopsis. We then present the resulting distribution pattern of CF in the shoots.

The symplastic tracer 5(6)-carboxyfluorescein diacetate (CFDA) has been widely applied in living plants to demonstrate the intercellular connection, ...

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.

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

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Biology

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Automated Stomatal Response Measurement to Bacterial Invasion in Arabidopsis

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake for photosynthesis and transpirational water loss but also for restricting bacterial invasion. While plants close stomata upon recognition of microbes, pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000 (Pto), reopen the closed stomata to gain access into the leaf interior. In conventional assays for assessing stomatal responses to bacterial invasion, leaf epidermal peels, leaf discs, or detached leaves are floated on bacterial suspension, and then stomata are observed under a microscope followed by manual measurement of stomatal aperture. However, these assays are cumbersome and may not reflect stomatal responses to natural bacterial invasion in a leaf attached to the plant. Recently, a portable imaging device was developed that can observe stomata by pinching a leaf without detaching it from the plant, together with a deep learning-based image analysis pipeline designed to automatically measure stomatal aperture from leaf images captured by the device. Here, building on these technical advances, a new method to assess stomatal responses to bacterial invasion in Arabidopsis thaliana is introduced. This method consists of three simple steps: spray inoculation of Pto mimicking natural infection processes, direct observation of stomata on a leaf of the Pto-inoculated plant using the portable imaging device, and automated measurement of stomatal aperture by the image analysis pipeline. This method was successfully used to demonstrate stomatal closure and reopening during Pto invasion under conditions that closely mimic the natural plant-bacteria interaction.

Author Spotlight: Advancing Stomatal Research with Automated Aperture Measurement

Here, we present a simple method for direct observation and automated measurement of stomatal responses to bacterial invasion in Arabidopsis thaliana. This method leverages a portable stomatal imaging device, together with an image analysis pipeline designed for leaf images captured by the device.

Stomata are microscopic pores found in the plant leaf epidermis. Regulation of stomatal aperture is pivotal not only for balancing carbon dioxide uptake ...

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called thigmomorphogenesis. In past decades, several signaling components have been identified and reported for being involved in the mechanotransduction (e.g., calcium ion binding proteins and jasmonic acid biosynthesis enzymes). However, the relatively slow pace of research in the study of force signaling or thigmomorphogenesis is largely attributed to two reasons: the requirement for laborious human hand-manipulated touch induction of thigmomorphogenesis and the force strength errors associated with people&#8217;s hand-touch. To enhance the efficiency of external force loading on a plant organism, an automatic touch-force loading machine was built. This robotic arm-driven hair brush touches provide a labor-saving and easily repeatable touch-force simulation, unlimited rounds of touch repetition and adjustable touch strength. This hair touch-force loading machine can be used for both large scale screening of touch-force signaling mutants and the phenomics study of plant thigmomorphogenesis. In addition, touch materials such as human hair, can be replaced with other natural materials like animal hair, silk threads and cotton fibers. The automated moving arms on the machine may be equipped with water sprinkling nozzles and air blowers to mimic the natural forces of rain drops and wind, respectively. By using this automatic hair touch-force loading machine in combination with the hand-performed cotton swab touching, we have investigated the touch response of two force signaling mutants, MAP KINASE KINASE 1 (MKK1) and MKK2 plants. The phenomes of the touch-force loaded wild type plants and two mutants were evaluated statistically. They have exhibited significant differences in touch response.

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

A gentle touch-force loading machine is built from human hair brushes, robotic arms and a controller. The hair brushes are driven by robotic arms installed on the machine and move periodically to apply touch-force on plants. The strength of machine-driven hair touches is comparable to that of manually applied touches.

Plants responding to both intracellular and extracellular mechanical stimulations (or force signals) and develop special morphological changes, a called ...

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental step required towards this end. Thus, it is paramount to establish high-throughput Arabidopsis seed surface sterilization methods to handle tens to hundreds of samples (e.g., transgenic lines, ecotypes, or mutants) at once. A seed surface sterilization method based on the efficient elimination of liquid in tubes with a homemade suction device constructed from a common vacuum pump is presented in this study. By dramatically reducing labor-intensive hands-on time with this method handling several hundreds of samples in one day is possible with little effort. Series time-course analyses further indicated a highly flexible time range of surface sterilization by maintaining high germination rates. This method could be easily adapted for surface sterilization of other kinds of small seeds with simple customization of the suction device according to the seed size, and the speed desired to eliminate the liquid.

A high-throughput protocol for the surface sterilization of Arabidopsisthaliana (Arabidopsis) seeds is provided, optimizing the liquid handling steps with a simple suction device constructed with a vacuum pump. Hundreds of seed samples can be surface-sterilized in one day.

Arabidopsis is by far the plant model species most widely used for functional studies. The surface sterilization of Arabidopsis seeds is a fundamental ...

High-Resolution Imaging of Chlorophyll Biosynthesis in Arabidopsis Seedling

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Author Spotlight: Non-Invasive High-Resolution Measurement of Chlorophyll Synthesis During De-Etiolation

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly ...

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.

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 ...

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

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Chapters in this video

Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants

Shootward Movement of CFDA Tracer Loaded in the Bottom Sink Tissues of Arabidopsis

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

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::β-glucuronidase Reporter Gene Expression

Detection of Histone Modifications in Plant Leaves

Direct Observation and Automated Measurement of Stomatal Responses to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis thaliana

A Labor-saving and Repeatable Touch-force Signaling Mutant Screen Protocol for the Study of Thigmomorphogenesis of a Model Plant Arabidopsis thaliana

High-throughput, Robust and Highly Time-flexible Method for Surface Sterilization of Arabidopsis Seeds

Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Measuring Immune-Induced Inhibition of Seedling Growth