We thank Dr. Shahid Mukhtar for critiquing the manuscript and Dr. Xinnian Dong for the sample data analysis file. This work is supported by a NSF-CAREER award (IOS-1350244) to KPM and the UAB Biology Department.

The following text describes a stepwise protocol to perform optimized Psm ES4326 syringe infiltration assay in Arabidopsis. Major procedures of this assay are represented in a simplified flowchart (Figure 1).
1. Plant Growth Conditions
<ol>
	<li>Sow seeds
	<ol>
		<li>Prepare 2 pots (4 in&#160;diameter, 3.75 in&#160;tall) loosely filled with soil and water pots by soaking them from the bottom O/N before draining the excess water.</li>
		<li>Sow 50-100 Arabidopsis seeds, wild-type Col-0 or npr1-1 mutant, on each pot with a folded 70 mm weighing sheet or other paper.</li>
		<li>Cover the pot with a water-sprayed transparent dome to increase the relative humidity to 80-90% (Figure 2A).</li>
	</ol>
	</li>
	<li>Incubate the pot at 4 &#176;C for 72 hr to allow for complete stratification and synchronous germination.</li>
	<li>Transfer the pot to the standard growth conditions (12 hr light/12 hr dark, 21 &#176;C, light intensity 100 &#181;mol/m2/sec, relative humidity 40%) to allow the seed to germinate. Crack the dome to generate a 2-3 in&#160;opening immediately after transfer to the growth room, which will help avoid water condensation.</li>
	<li>When the first true leaf reaches the size of 2-3 mm in length, transplant the seedlings from the pot to a 72-wells flat filled with soil.
	<ol>
		<li>Use a finger or a 1 ml&#160;pipette tip to create a 1-in&#160;deep depression in the middle of the soil surface on the flat.</li>
		<li>Gently separate individual seedlings with as little root damage as possible. Carefully pick up seedlings that have intact roots with forceps.</li>
		<li>Place a single separated seedling together with the adhering soil clump to minimize transplantation shock into the depression and gently pat down the surrounding soil to fill the depression. Discard the extra seedlings and soil into biohazard waste container and dispose in accordance with the local biohazard waste disposal guidelines.</li>
		<li>Water transferred seedlings from the top and completely cover the flat with a water-sprayed transparent dome to maintain 80-90% humidity. Keep the dome on for 3 days, then crack the dome in the morning of day 4 and completely remove it by the end of that day. 
		Note: Sowing seeds can be alternatively performed by stratifying seeds in 1.5 ml&#160;centrifuge tubes containing 1 ml&#160;0.1% agar for 48 hr at 4 &#176;C followed by transferring 2-3 seeds with a glass pipette onto a 72-wells flat filled with soil. Flats need to be covered with a transparent dome that can be removed one week after the germination. Extra seedlings can be removed to leave only one seedling per well.</li>
	</ol>
	</li>
	<li>Water plants every two days by soaking flats in 1-in&#160;water for 20 min, then drain the excess water. 
	Note: The time interval for watering plants depends on the growth room humidity and needs to be determined based on continuous observation of user&#8217;s plant growth facility. Check plants daily to make sure they are well watered. If only few spots are drying, water those plants from the top with a squirt bottle.</li>
	<li>Perform pathogen infection assays (Step 3-5) on plants that are at or near developmental stage # 3.50 (when rosette size is 50% final size), which corresponds to 3-5 weeks old depending on growth conditions (photoperiod, temperature). Do not infect plants after inflorescence emergence (stage # 5) due to onset of age-related resistance38,39.</li>
</ol>
2. Preparation of Culture Media and Plates
<ol>
	<li>Make 1 L of King&#8217;s B (KB) liquid medium. Gently stir 20 g proteose peptone and 2 g potassium phosphate dibasic trihydrate using a magnetic stir bar with 1,000 ml&#160;deionized H2O until there is no visible pellet. Aliquot 100 ml&#160;into 150 ml&#160;glass bottles and autoclave on a 20-min liquid cycle.</li>
	<li>Prepare culture plates with KB solid medium.
	<ol>
		<li>Gently stir 20 g Proteose Peptone, 2 g Potassium Phosphate Dibasic Trihydrate and 15 g Agar with 1,000 ml&#160;deionized H2O. Autoclave.</li>
		<li>Cool down the medium on a magnetic stir plate until it is cool to touch to minimize future condensation and avoid degradation of antibiotics. Add 18 ml&#160;sterile 80% Glycerol and 5 ml&#160;sterile 1 M MgSO4 to the medium. Given that Psm ES4326 carries resistance against streptomycin, add streptomycin into the cooled media to a final concentration of 50 &#181;g ml&#8722;1.</li>
		<li>Pour the plates. Prepare two different sizes of KB media plates: 100 mm x 15 mm and 150 mm x 15 mm Petri dishes. The smaller Petri dish containing medium serves as the primary bacteria culture plate, and the larger Petri dish serves as bacteria counting plate. 
		Note: For the bacteria counting plates, rectangular-shaped plates can be used to reduce the consumables cost since twice as many treatments can be plotted per plate compared to traditional round plates.
		<ol>
			<li>Pour the plates prior to the infection experiment. Store KB medium plates at 4 &#176;C for up to 2-3 months since streptomycin sulfate gradually loses the antibiotic activity40.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. Enhanced Disease Susceptibility (EDS)
<ol>
	<li>Day 1 - Streak bacteria on plate
	<ol>
		<li>Two days before the EDS assay, streak Psm ES4326 from -80 &#176;C glycerol stock on a KB-Strep bacterial culture plate and incubate at 28 &#176;C for 24-48 hr.</li>
	</ol>
	</li>
	<li>Day 2 - initiate liquid culture and water plants
	<ol>
		<li>Initiate the liquid culture in a sterile test tube with 4 ml&#160;liquid KB medium containing 50 &#181;g ml&#8722;1 streptomycin and shake at 250 rpm, 28 &#176;C O/N. 
		Note: For EDS infection assay, using 6 plants per genotype is recommended. For STI and MTI assays, 6 plants per genotype per treatment are recommended. For the bacteria inoculum, it is recommended to use a fresh liquid culture with optical density at &#955;600nm between 0.3 and 0.6.</li>
		<li>Mark the petioles of leaves number 5 and 6 with a blunt-end waterproof marker for easy identification of infected tissue at sampling (Figure 1). To enhance the opening of stomata and facilitate the entrance of pathogen solution into the leaf, water the plants well by soaking the flat from the bottom for 20 min, then drain the excess water. 
		Note: Alternatively, cover the flat with a transparent dome and soak it in water for 2-4 hr to increase the stomatal opening.</li>
		<li>Day 3 - Pathogen dilution and syringe infiltration 
		Note: Pathogen infection during morning hours is optimal for pathogen proliferation and development of the most pronounced disease symptoms on susceptible genotypes. Make every effort to keep the infection timing consistent to eliminate the effect of circadian rhythms and diurnal gene regulation41,42, which helps reduce the variation among experimental replications.
		<ol>
			<li>Pellet the bacterial culture in a microcentrifuge 1.5 ml&#160;tube at 9,600 x g for 2 min at RT and discard the supernatant. Resuspend the bacterial pellet with 1 ml&#160;of sterile 10 mM MgCl2. 
			Note: Magnesium cations can enhance the motility and adhesion of P. syringae43. Alternatively, use MgSO4 at the same concentration. Sterile water is an acceptable substitute for the Mg salt solutions.</li>
			<li>Dilute the bacteria suspension with 9 ml&#160;of 10 mM MgCl2 in a 50 ml&#160;centrifuge tube and measure the optical density (OD) of bacteria with a spectrophotometer at &#955;=600 nm. Dilute the bacteria with 10 mM MgCl2 to the final OD600nm=0.0002 for EDS infection assay.</li>
			<li>Infiltrate the leaf with a 1 ml&#160;blunt end needleless syringe (commonly known as the insulin syringe) that contains the diluted bacterial solution.</li>
			<li>Do not fill the syringe up to its full capacity. Fill it up with 0.5-0.6 ml&#160;to allow for a much better control during the infiltration process.</li>
			<li>Expose the lower surface of the leaf on the top of index finger, and then gently adjust the leaf position with the help of thumb. Position the syringe vertically against the leaf surface to ensure that pressure is evenly distributed. Try to avoid the midrib area during the infiltration to reduce leaf damage.</li>
			<li>Slowly push the plunger to infiltrate the bacterial solution; liquid entry into the leaf mesophyll will be visualized as indicated by the darker leaf color. Attempt to infiltrate the entire surface of the leaf. If this is not accomplished in a single attempt, choose another infiltration spot and repeat the actions described above until the entire leaf surface is covered.</li>
			<li>Once completed, gently blot the leaf with absorbent tissue to remove the extra pathogen solution. Visually inspect the infected leaf tissue for damage. 
			Note: The circular syringe impression should not be visible after the infiltration is complete.</li>
			<li>Leave the infiltrated plants to dry for 1-2 hr before returning them into their original growth conditions. Next, spray a clear dome with water and cover the infected plants for 2 hr, then crack the dome to generate a 2-3 in&#160;opening and leave it on throughout the remainder of the infection experiment. 
			Note: Since P. syringae is not an airborne pathogen, there is no risk of spreading the infectious agent to other plants grown in the same facility. However, as an added precaution, avoid physical contact between infected plants and other experimental plants located nearby. Covering the flat with a transparent dome will increase the pathogen virulence and accelerate disease progression. The need for this step and optimal duration of the covering period needs to be determined based on the humidity conditions of the user&#8217;s plant growth facility.</li>
		</ol>
		</li>
		<li>Day 5 - Pre-dry the media plates
		<ol>
			<li>Take the 150 mm x 15 mm KB plates out of the cold storage unit and dry any pre-existing water condensation on the plate. Dry plates by keeping them at RT for roughly 24 hr. To speed up this process, place them in a laminar flow hood with their lids cracked for 30-60 min. 
			Note: This step is critical for the formation of circular droplet on the surface of the plate in the next step.</li>
		</ol>
		</li>
		<li>Day 6 - Quantifying the pathogen growth 
		Note: The following pathogen quantification procedure may be performed at earlier time points after the infection to confirm equal amounts of bacteria are delivered into the leaf, especially when plants have altered leaf morphology.
		<ol>
			<li>Process the infected tissue after the emergence of chlorosis indicated by the yellowing of the infected tissue in the susceptible genotypes, but before the development of necrotic lesions (Figure 2B). 
			Note: Suggested sampling time is three days after the pathogen inoculation. However, since pathogen growth depends on a number of environmental factors, the length of incubation may vary within a range of 2 &#189;-3 &#189; days and needs to be determined by careful observations of the infection progression in user&#8217;s plant growth facility.
			<ol>
				<li>Prepare 6 grinding tubes for each genotype (Figure 1). Place one stainless steel grinding ball and add 500 &#181;l&#160;sterile 10 mM MgCl2 into the each tube.</li>
				<li>Detach the infected leaf from the plant and punch a leaf disc with a 1-hole paper punch (Figure 1). 
				Note: To minimize the sampling error, try to punch each sampled leaf at the same position. Sampling of the leaf disc from the top of the leaf is recommended.</li>
				<li>Randomly place 2 leaf discs (from two different plants) into each grinding tube using forceps.</li>
				<li>Seal the tube and homogenize the tissue with a high-throughput homogenizer at maximum speed (1,600 strokes per minute) for 10 min. Repeat this process if needed until tissue is well homogenized and the solutions turn green due to chlorophyll release from the infected leaves.</li>
			</ol>
			</li>
			<li>While waiting for the homogenization, fill the first 6 rows of a 96-well culture plate with 180 &#181;l&#160;10 mM MgCl2 using a multi-channel pipette and a pipetting reservoir.</li>
			<li>After grinding, transfer 20 &#181;l&#160;of ground tissue suspension into the first row of the 96-well plate and mix by repeated pipetting the liquid up and down. If small fragments of tissue clog the tip, clip it by 2-3 mm to help acquire the correct volume of the solution.
			<ol>
				<li>To provide enough space for the droplet on the top of the plate, space the tissue from different genotypes in alternative rows (Figure 1). To prepare a ten-fold serial dilution, transfer 20 &#181;l&#160;liquid into the second row and repeat this procedure until the sixth dilution.</li>
			</ol>
			</li>
			<li>Transfer 20 &#181;l&#160;of the solution from the 96-well plate onto the 150 mm x 15 mm KB plate using divided pipette tips (Figure 1). Work from the most dilute suspension to the most concentrated. 
			Note: Proceeding from most diluted to most concentrated makes it unnecessary to change pipette tips between the dilutions. If the plate is well pre-dried, the transferred droplet should stay intact on the top of the medium until absorbed (usually 15-30 min). Drying time varies with the RT and humidity.</li>
			<li>Dry the plate at RT with lid cracked. Once no more liquid can be observed on the plate surface, close the lid, invert and incubate the plate at RT or a 28 &#176;C incubator.</li>
			<li>Incubate plates for 40-60 hr until the colonies become visible. Confirm that the growth on the plates reflects the predictable 10-fold drop in colony forming units (cfu) (Figure 2C). Count the bacteria before they overgrow and colonies fuse. Determine the number of bacteria in the lowest dilution that does not have overlapping colonies. Usually, the preferred dilution to be counted will contain between 10-50 colonies. 
			Note: Variation may be present among technical replicates; therefore, the lowest dilution for each replicate should be determined separately.</li>
			<li>To calculate the levels of bacterial proliferation, document the number of the row (R) as well as the number of the bacteria within each technical replicate (T) in writing. Determine the number of colony forming unit &#8211; cfu/leaf disc through the formula: cfu/leaf disc = (T &#215; 10R / 20) &#215; 500 / 2</li>
			<li>Type the data in the following spreadsheet template to produce a graph (Figure 1) (for spreadsheet data analysis template file, see Table 2). Each data point is represented as the mean of six technical replicates on a logarithmic scale. Error bars represent 95% confidence interval of the mean (n = 6). 
			Note: The calculation of 95% confidence intervals needs to be adjusted based on the number of technical replicates.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
4. SA-Triggered Immunity (STI)
<ol>
	<li>Day 1: Follow the same procedure as described in step 3.1.</li>
	<li>Day 2: In addition to watering the plants and initiating the liquid bacterial culture (Step 3.2), induce resistance by external application of SA derivative Sodium Salicylate21. 
	Note: Pure SA has poor water solubility and requires prior pH adjusting, thus it&#8217;s not recommended for this assay.
	<ol>
		<li>To induce SA-mediated defenses against P. syringae and prime the plants for future infection21,24, spray plants with a fine mist of 1 mM Sodium Salicylate or H2O (as mock) 16-24 h before the pathogen infection.</li>
	</ol>
	</li>
	<li>Day 3: Prepare pathogen dilution to OD600nm=0.001 and push the bacteria into the entire leaf surface by syringe infiltration (Step 3.3).</li>
	<li>Day 6: For pathogen quantification and counting process, follow the procedure as described for the EDS assay (Steps 3.4 and 3.5). Plate and count the H2O and Sodium Salicylate &#8211; pretreated samples separately. For wild-type Arabidopsis, a 1-2 log reduction in pathogen growth is expected in the Sodium Salicylate &#8211; pretreated plants.</li>
</ol>
5. MAMP-Triggered Immunity (MTI)

Note: In this assay, we use Arabidopsis wild-type Col-0 and mutant fls2 and efr plants. FLS2 and EFR are plasma membrane-localized Pattern Recognition Receptors (PRRs) that can recognize flg22 and elf18, respectively9,10. Loss of each PRR results in the insensitivity to the specific type of MAMP, which is indicated by the unaltered pathogen growth in the mutant following external MAMP pre-treatment and syringe infiltration with Psm ES4326.
<ol>
	<li>Day 1 and 2: Follow the same procedure as described in Step 3.1 and 3.2.</li>
	<li>Day 3: MAMP pre-treatment and Pathogen infection
	<ol>
		<li>To establish the MAMP-Triggered Immunity, prepare 1 &#181;M solution of flagellin epitope flg22 or EF-Tu epitope elf18 and syringe-infiltrate it into the entire leaf surface 4 h before the pathogen infection (Steps 3.3.3-3.3.6). This will allow for induction of MAMP-mediated defenses against P. syringae and prime the plants for future infection6,37. 
		Note: Publicly available microarray data revealed the maximum transcriptional reprogramming of MAMP-responsive genes happens 4 h after flg22 or elf18 treatment37,44. Thus, 4 hr is the recommended duration of the MAMP pre-treatment that results in achieving a clear difference in the pathogen growth between MAMP-treated Col-0 and fls2 and efr mutants.</li>
		<li>Remove the extra MAMP solution with absorbent tissue and leave the plants uncovered to speed the liquid absorption process within the leaf. To account for the wounding effect from syringe pressure infiltration, infiltrate H2O into the leaves of control plants.</li>
		<li>Prepare pathogen dilution to OD600nm=0.001 and push the bacteria into the entire leaf surface of MAMP/H2O pre-treated leaf by syringe infiltration (Step 3.3).</li>
	</ol>
	</li>
	<li>Day 6: For pathogen quantification and counting process, follow the procedure as described in Step 3.4 and 3.5. Plate and count the H2O and MAMP&#8211;pretreated samples separately. In wild-type Arabidopsis, a 1-2 log reduction in pathogen growth is expected in the MAMP&#8211;pretreated plants, with somewhat stronger reduction mediated by flg22 compared to elf18.</li>
</ol>

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<li>Wu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Arabidopsis+NPR1+protein+is+a+receptor+for+the+plant+defense+hormone+salicylic+acid%5BTitle%5D)+AND+%22Cell+Re%22%5BJournal%5D)">The Arabidopsis NPR1 protein is a receptor for the plant defense hormone salicylic acid.</a> Cell Re. 1, 639-647 (2012).</li>
<li>Smith, J. M., Heese, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Rapid+bioassay+to+measure+early+reactive+oxygen+species+production+in+Arabidopsis+leave+tissue+in+response+to+living+Pseudomonas+syringae%5BTitle%5D)+AND+%22Plant+Method%22%5BJournal%5D)">Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave tissue in response to living Pseudomonas syringae.</a> Plant Method. 10, 6(2014).</li>
<li>Nomura, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+bacterial+virulence+protein+suppresses+host+innate+immunity+to+cause+plant+disease%5BTitle%5D)+AND+%22Scienc%22%5BJournal%5D)">A bacterial virulence protein suppresses host innate immunity to cause plant disease.</a> Scienc. 313, 220-223 (2006).</li>
<li>Katagiri, F., Thilmony, R., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Arabidopsis+thaliana-Pseudomonas+syringae+interaction%5BTitle%5D)+AND+%22The+Arabidopsis+book%2FAmerican+Society+of+Plant+Biologist%22%5BJournal%5D)">The Arabidopsis thaliana-Pseudomonas syringae interaction.</a> The Arabidopsis book/American Society of Plant Biologist. 1, (2002).</li>
<li>Sawada, H., Suzuki, F., Matsuda, I., Saitou, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phylogenetic+analysis+of+Pseudomonas+syringae+pathovars+suggests+the+horizontal+gene+transfer+of+argK+and+the+evolutionary+stability+of+hrp+gene+cluster%5BTitle%5D)+AND+%22J+Mol+Evo%22%5BJournal%5D)">Phylogenetic analysis of Pseudomonas syringae pathovars suggests the horizontal gene transfer of argK and the evolutionary stability of hrp gene cluster.</a> J Mol Evo. 49, 627-644 (1999).</li>
<li>Xin, X. F., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+syringae+pv.+tomato+DC3000%3A+a+model+pathogen+for+probing+disease+susceptibility+and+hormone+signaling+in+plants%5BTitle%5D)+AND+%22Annu+Rev+Phytopatho%22%5BJournal%5D)">Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants.</a> Annu Rev Phytopatho. 51, 473-498 (2013).</li>
<li>Dong, X., Mindrinos, M., Davis, K. R., Ausubel, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induction+of+Arabidopsis+defense+genes+by+virulent+and+avirulent+Pseudomonas+syringae+strains+and+by+a+cloned+avirulence+gene%5BTitle%5D)+AND+%22Plant+Cel%22%5BJournal%5D)">Induction of Arabidopsis defense genes by virulent and avirulent Pseudomonas syringae strains and by a cloned avirulence gene.</a> Plant Cel. 3, 61-72 (1991).</li>
<li>Mackey, D., Holt 3rd, B. F., Wiig, A., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((RIN4+interacts+with+Pseudomonas+syringae+type+III+effector+molecules+and+is+required+for+RPM1-mediated+resistance+in+Arabidopsis%5BTitle%5D)+AND+%22Cel%22%5BJournal%5D)">RIN4 interacts with Pseudomonas syringae type III effector molecules and is required for RPM1-mediated resistance in Arabidopsis.</a> Cel. 108, 743-754 (2002).</li>
<li>Tornero, P., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+high%E2%80%90throughput+method+for+quantifying+growth+of+phytopathogenic+bacteria+in+Arabidopsis+thaliana%5BTitle%5D)+AND+%22The+Plant+Journa%22%5BJournal%5D)">A high‐throughput method for quantifying growth of phytopathogenic bacteria in Arabidopsis thaliana.</a> The Plant Journa. 28, 475-481 (2001).</li>
<li>Ishiga, Y., Ishiga, T., Uppalapati, S. R., Mysore, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Arabidopsis+seedling+flood-inoculation+technique%3A+a+rapid+and+reliable+assay+for+studying+plant-bacterial+interactions%5BTitle%5D)+AND+%22Plant+Method%22%5BJournal%5D)">Arabidopsis seedling flood-inoculation technique: a rapid and reliable assay for studying plant-bacterial interactions.</a> Plant Method. 7, 32(2011).</li>
<li>Zheng, X. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coronatine+promotes+Pseudomonas+syringae+virulence+in+plants+by+activating+a+signaling+cascade+that+inhibits+salicylic+acid+accumulation%5BTitle%5D)+AND+%22Cell+Host+Microb%22%5BJournal%5D)">Coronatine promotes Pseudomonas syringae virulence in plants by activating a signaling cascade that inhibits salicylic acid accumulation.</a> Cell Host Microb. 11, 587-596 (2012).</li>
<li>Pajerowska-Mukhtar, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+HSF-like+transcription+factor+TBF1+is+a+major+molecular+switch+for+plant+growth-to-defense+transition%5BTitle%5D)+AND+%22Curr+Bio%22%5BJournal%5D)">The HSF-like transcription factor TBF1 is a major molecular switch for plant growth-to-defense transition.</a> Curr Bio. 22, 103-112 (2012).</li>
<li>Rusterucci, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Age-related+resistance+to+Pseudomonas+syringae+pv.+tomato+is+associated+with+the+transition+to+flowering+in+Arabidopsis+and+is+effective+against+Peronospora+parasitica%5BTitle%5D)+AND+%22Physiological+and+molecular+plant+patholog%22%5BJournal%5D)">Age-related resistance to Pseudomonas syringae pv. tomato is associated with the transition to flowering in Arabidopsis and is effective against Peronospora parasitica.</a> Physiological and molecular plant patholog. 66, 222-231 (2005).</li>
<li>Boyes, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+stage%E2%80%93based+phenotypic+analysis+of+Arabidopsis+a+model+for+high+throughput+functional+genomics+in+plants%5BTitle%5D)+AND+%22The+Plant+Cell+Onlin%22%5BJournal%5D)">Growth stage–based phenotypic analysis of Arabidopsis a model for high throughput functional genomics in plants.</a> The Plant Cell Onlin. 13, 1499-1510 (2001).</li>
<li>Regna, P. P., Wasselle, L. A., Solomons, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+stability+of+streptomycin%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistr%22%5BJournal%5D)">The stability of streptomycin.</a> Journal of Biological Chemistr. 165, 631-638 (1946).</li>
<li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Timing+of+plant+immune+responses+by+a+central+circadian+regulator%5BTitle%5D)+AND+%22Natur%22%5BJournal%5D)">Timing of plant immune responses by a central circadian regulator.</a> Natur. 470, 110-114 (2011).</li>
<li>Hua, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Modulation+of+plant+immunity+by+light%2C+circadian+rhythm%2C+and+temperature%5BTitle%5D)+AND+%22Current+opinion+in+plant+biolog%22%5BJournal%5D)">Modulation of plant immunity by light, circadian rhythm, and temperature.</a> Current opinion in plant biolog. 16, 406-413 (2013).</li>
<li>Cuppels, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemotaxis+by+Pseudomonas+syringae+pv.+tomato%5BTitle%5D)+AND+%22Appl+Environ+Microbio%22%5BJournal%5D)">Chemotaxis by Pseudomonas syringae pv. tomato.</a> Appl Environ Microbio. 54, 629-632 (1988).</li>
<li>Qutob, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phytotoxicity+and+innate+immune+responses+induced+by+Nep1-like+proteins%5BTitle%5D)+AND+%22The+Plant+Cell+Onlin%22%5BJournal%5D)">Phytotoxicity and innate immune responses induced by Nep1-like proteins.</a> The Plant Cell Onlin. 18, 3721-3744 (2006).</li>
<li>Pimentel, D., Lach, L., Zuniga, R., Morrison, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Environmental+and+economic+costs+of+nonindigenous+species+in+the+United+States%5BTitle%5D)+AND+%22BioScienc%22%5BJournal%5D)">Environmental and economic costs of nonindigenous species in the United States.</a> BioScienc. 50, 53-65 (2000).</li>
<li>Zeng, W., Melotto, M., He, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+stomata%3A+a+checkpoint+of+host+immunity+and+pathogen+virulence%5BTitle%5D)+AND+%22Current+opinion+in+biotechnolog%22%5BJournal%5D)">Plant stomata: a checkpoint of host immunity and pathogen virulence.</a> Current opinion in biotechnolog. 21, 599-603 (2010).</li>
<li>Ton, J., Flors, V., Mauch-Mani, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+multifaceted+role+of+ABA+in+disease+resistance%5BTitle%5D)+AND+%22Trends+in+plant+scienc%22%5BJournal%5D)">The multifaceted role of ABA in disease resistance.</a> Trends in plant scienc. 14, 310-317 (2009).</li>
<li>Mahajan, S., Tuteja, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cold+salinity+and+drought+stresses%3A+an+overview%5BTitle%5D)+AND+%22Archives+of+biochemistry+and+biophysic%22%5BJournal%5D)">Cold salinity and drought stresses: an overview.</a> Archives of biochemistry and biophysic. 444, 139-158 (2005).</li>
<li>León, J., Rojo, E., Sánchez‐Serrano, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Wound+signalling+in+plants%5BTitle%5D)+AND+%22Journal+of+Experimental+Botan%22%5BJournal%5D)">Wound signalling in plants.</a> Journal of Experimental Botan. 52, 1-9 (2001).</li>
</ol>

The authors have nothing to disclose.

With decreasing available farmland and increasing population, researchers around the world are challenged with pressing needs for crop improvement. The yield can be greatly influenced by various biotic and abiotic stresses. Among them, pathogen infection is one of the leading causes of crop yield reduction, responsible for approximately 12% losses in the U.S. alone45. To resolve this issue, massive research has been conducted in the model Arabidopsis - P. syringae pathosystem to comprehensively charac...

Due to their sessile nature, plants are constantly threatened by a plethora of pathogens exhibiting various lifestyles and nutritional strategies1. To a first approximation, biotrophic pathogens maintain their host alive to retrieve nutrients, while necrotrophic pathogens actively secret toxins and enzymes to kill host tissue and feed on the dead cells1. Another group of pathogens, termed hemibiotrophs, begins their infection course with the biotrophic stage and shifts to the necrotrophic stage upon reaching a certain threshold of pathogen accumulation2. In order to effectively defend themselves against these microorganisms, plants have evolved a complicated innate immune system equipped with multiple surveillance mechanisms to detect the pathogen attack and trigger localized programmed cell death3 as well as Systemic Acquired Resistance (SAR)4. Current research is focused on characterizing the essential signaling components and cross-talks within the plant immune system5.

As proposed in the &#8220;Zig-Zag&#8221; model5, the first layer of the plant innate immune response requires the presence of plasma membrane-localized Pattern Recognition Receptors (PRRs) to detect the invasion of a microbe. PRRs are able to recognize Microbe-Associated Molecular Patterns (MAMPs) and establish MAMP-Triggered Immunity (MTI)6. Besides inducing a transcriptional upregulation of genes encoding antimicrobial PR proteins7, MTI leads to a variety of events that arrest pathogen growth, including the production of Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), deposition of callose to the cell wall as well as the activation of multiple kinase signaling pathways8.
Until now, several MAMPs have been identified to trigger MTI in Arabidopsis, including bacterial flg229 (a 22 amino acid fragment derived from flagellin), elf1810 (18 amino acids from the bacterial translation elongation factor Tu) and a structural cell wall component peptidoglycans11. To establish a successful infection, some specialized pathogens have evolved the ability to secret virulence effector proteins into the intracellular or intercellular spaces, and consequently repress MTI and trigger Effector-Triggered Susceptibility (ETS)12,13. For instance, virulence effectors can inactivate Mitogen-Activated Protein Kinase (MAPK) phosphorylation cascades of MTI to induce the disease development within the infected tissue14-16. During the dynamic co-evolution between hosts and pathogens, plants also developed the counterattack strategy to recognize the effector proteins and attenuate the pathogen virulence molecules17. This direct or indirect effector recognition is mediated by disease resistance (R) proteins18. Most of them are members of NB-LRR (Nucleotide Binding and Leucine-Rich Repeats) family19. The perception of an avirulent effector by an R protein elicits a stronger and broader immune response characterized as Effector-Triggered Immunity (ETI)20. Besides inducing the expression of defense genes21 and the production of defense metabolites22, ETI often leads to a rapid localized programmed cell death known as Hypersensitive Response (HR) to restrict the pathogen from spreading into the adjacent tissue3.
In addition to the localized programmed cell death23, plants are capable of initiating a long-term and system-wide immune response termed Systemic Acquired Resistance (SAR)4. Upon challenge with a biotrophic pathogen, plant cells trigger the biosynthesis and accumulation of an endogenous phytohormone salicylic acid (SA) and PR proteins in both local and systemic tissues24. Through this process, a heightened state of preparedness is achieved in the uninfected leaves that allows for mounting faster defense responses during a subsequent infection by a broad spectrum of pathogens24. SA and its synthetic analogs such as benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) and 2,6-dichloroisonicotinic acid (INA) are capable of chemically inducing Salicylic Acid (SA)-Triggered Immunity (STI) upon external application24. Nonexpressor of Pathogenesis-Related genes 1 (NPR1) is proposed to be one of the SA receptors and functions as a major transcriptional regulator during SA-mediated defense response in both local and systemic tissues21,25,26. It has been conclusively demonstrated that NPR1 is required for SAR establishment and the loss of NPR1 leads to dramatic susceptibility towards Pseudomonas syringae25.
To extensively characterize the molecular contribution of plant components in the plant-pathogen interactions, multiple bioassays have been developed to measure specific defense events, including ROS burst27, callose deposition28, defense genes expression and accumulation of their protein products21. While these individual assays can provide insights into a specific form of the plant immune response, none of them, however, are able to represent the complete defense response on the whole plant level. Conversely, quantification of pathogen growth after infection provides an overall estimation of immune response at the organismal level. Therefore, the development and optimization of a precise and highly standardized pathogen inoculation assay is critical to fuel up the research and discoveries on the Arabidopsis immune responses.
Pseudomonas syringae, a Gram-negative bacterium, was identified as a phytopathogen capable of causing disease in a range of plant hosts including Arabidopsis29. As the model plant-pathogen system, the Arabidopsis - P. syringae interaction has been widely applied to understand the molecular mechanisms underlying plant defense responses29. Until now, over 50 P. syringae pathovars have been identified based on their ability to infect different plant species30 . P. syringae pv. tomato DC3000(Pst DC3000)31and P. syringae pv. maculicola ES4326 (Psm ES4326)32 are the two most widely used and extensively characterized virulent strains. Aside from being recognized by the plant and triggering the MTI response, Pst DC3000 and Psm ES4326 are capable of secreting virulent effector proteins to suppress MTI and trigger ETS to favor the pathogen growth31,33.
To functionally dissect the interaction between Arabidopsis and P. syringae, multiple&#160;pathogen infection methods have been developed based on the pathogen delivery approach. For soil-grown plants, pathogen can be delivered by syringe infiltration, vacuum infiltration, dip inoculation and spray inoculation29,34. Recently, seedling flood-inoculation assay was developed to perform large-scale screens on tissue culture plates-grown young Arabidopsis plants35. Syringe infiltration, as one of the most commonly used approach, manually delivers the pathogen into the apoplast through the natural leaf openings termed stomata29. Through this approach, equal amounts of P. syringae can be infiltrated into the infected leaf and the strength of plant immune response is inversely correlated to the pathogen growth levels. Therefore, quantification of pathogen growth serves as an optimal approach to evaluate the immune function at the whole plant level. In addition, syringe infiltration can distinguish the local and systemic tissue, which can be applicable in characterizing the molecular mechanisms underlying SAR36.
In the following protocol, we describe an optimized syringe infiltration assay with Psm ES4326 to screen Arabidopsis mutants for enhanced disease susceptibility (EDS). This protocol will employ two Arabidopsis genotypes: a wild-type ecotype Columbia-0 (Col-0) plants (control) and npr1-1 loss-of-function&#160;mutants (hypersusceptible) that will be infected with virulent bacterial strain Psm ES432637. The npr1-1 mutant carries a point mutation within the ankyrin-repeat consensus sequence of the NPR1 molecule, which alters the highly conserved histidine to tyrosine and renders the protein non-functional25. In addition, a number of modifications of the syringe infiltration assay are described that allow quantification of defects in the specific layers of immune response, including MTI and STI.

<table><tbody><tr><td>MetroMix 360&amp;nbsp;</td><td>Grosouth</td><td>SNGMM360</td><td></td></tr><tr><td>Large pots</td><td>Grosouth</td><td>TEKUVCC10TC</td><td></td></tr><tr><td>12 x 6 Inserts</td><td>Grosouth</td><td>LM1206</td><td></td></tr><tr><td>11x 21 Flats with no holes</td><td>Grosouth</td><td>LM1020</td><td></td></tr><tr><td>11x 21 Flats with holes</td><td>Grosouth</td><td>LM1020H</td><td></td></tr><tr><td>Vinyl propagation domes</td><td>Grosouth</td><td>CW-221</td><td></td></tr><tr><td>Proteose Peptone</td><td>Fisher Scientific</td><td>DF0122-17-4</td><td></td></tr><tr><td>Potassium Phosphate Dibasic Trihydrate&amp;nbsp;</td><td>MP Biomedicals</td><td>151946</td><td></td></tr><tr><td>Agar&amp;nbsp;</td><td>Fisher Scientific</td><td>A360-500</td><td></td></tr><tr><td>Streptomycin sulfate</td><td>Bio Basic Inc</td><td>SB0494</td><td></td></tr><tr><td>100 x 15 mm Petri dishes</td><td>Fisher Scientific</td><td>FB0875713</td><td></td></tr><tr><td>150 x 15 mm Petri dishes</td><td>Fisher Scientific</td><td>R80150</td><td></td></tr><tr><td>Rectangular plate</td><td>Fisher Scientific</td><td>12-565-450&amp;nbsp;</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2 &lt;/sub&gt;Hexahydrate</td><td>Bio Basic Inc</td><td>MB0328</td><td></td></tr><tr><td>Glycerol</td><td>Bio Basic Inc</td><td>GB0232</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&amp;nbsp;&lt;/sub&gt;</td><td>Bio Basic Inc</td><td>MN1988</td><td></td></tr><tr><td>1 ml syringe</td><td>Fisher Scientific</td><td>NC9992493&amp;nbsp;</td><td></td></tr><tr><td>Kimwipe</td><td>Fisher Scientific</td><td>06-666-A</td><td></td></tr><tr><td>Grinding tubes&amp;nbsp;</td><td>Denville Scientific</td><td>B1257</td><td></td></tr><tr><td>Caps for grinding tubes</td><td>Denville Scientific</td><td>B1254</td><td></td></tr><tr><td>Stainless steel grinding ball</td><td>Fisher Scientific</td><td>2150</td><td></td></tr><tr><td>96-well plate&amp;nbsp;</td><td>Fisher Scientific</td><td>12-556-008</td><td></td></tr><tr><td>Sodium Salicylate</td><td>Sigma Aldrich</td><td>s3007-1kg</td><td></td></tr><tr><td>flg22 (QRLSTGSRINSAKDDAAGLQIA)</td><td>Genescript</td><td>Made to order</td><td></td></tr><tr><td>elf18 (Ac-SKEKFERTKPHVNVGTIG)</td><td>Genescript</td><td>Made to order</td><td></td></tr><tr><td>Hole puncher</td><td>Staples</td><td>146308</td><td></td></tr><tr><td>Biophotometer plus</td><td>Eppendorf</td><td>952000006</td><td></td></tr><tr><td>PowerGen High-Throughput Homogenizer</td><td>Fisher Scientific</td><td>02-215-503</td><td></td></tr><tr><td>Accu spin micro centrifuge</td><td>Fisher Scientific</td><td>13-100-675</td><td></td></tr><tr><td>Multichannel pipette (10-100 &amp;micro;l)</td><td>Eppendorf</td><td>3122 000.043</td><td></td></tr><tr><td>Multichannel pipette (30-300 &amp;micro;l)</td><td>Eppendorf</td><td>3122 000.060</td><td></td></tr><tr><td>Pipette (20&amp;micro;l)</td><td>Eppendorf</td><td>3120 000.038</td><td></td></tr><tr><td>Pipette tips</td><td>Fisher Scientific</td><td>3552-HR</td><td></td></tr><tr><td>Sharpie permanent marker</td><td>Staples</td><td>507130</td><td></td></tr><tr><td>1.5 ml tube</td><td>Eppendorf</td><td>22363204</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>08-890</td><td></td></tr></tbody></table>

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).

The protocol we describe here represents an optimized P. syringae syringe infiltration assay to quantitatively evaluate the immune response in Arabidopsis plants. As illustrated in Figure 1, the syringe infiltration of Psm ES4326 is followed by pathogen extraction and quantification via serial dilutions and colonies enumeration.
As described in Step 3 within the protocol text, Enhanced Disease Susceptibility (EDS) against Psm ES4326 can be a...

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Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem | Immunology and Infection | Video

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.

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 ...

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21.8K Views.  University of Alabama at Birmingham. 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.

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

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 ...

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.

We have developed a simple and reproducible protocol to access stomatal response to live bacteria. This method minimizes wounding and manipulation of the leaf as compared to the use of epidermal peels reported previously.

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

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.

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

Bioengineering

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.

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.

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 ...

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 ...

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins.
Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th &#946;-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th &#946;-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Fluorescent protein-based approaches to monitor effectors secreted by bacteria into host cells are challenging. This is due to the incompatibility between fluorescent proteins and the type-III secretion system. Here, an optimized split superfolder GFP system is used for visualization of effectors secreted by bacteria into the host plant cell.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the ...

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic bacteria of special relevance for a wide number of hosts. P. syringae enters the plant from the leaf surface and multiplies rapidly within the apoplast, forming microcolonies that occupy the intercellular space. The constitutive expression of fluorescent proteins by the bacteria allows for visualization of the microcolonies and monitoring of the development of the infection at the microscopic level. Recent advances in single-cell analysis have revealed the large complexity reached by clonal isogenic bacterial populations. This complexity, referred to as phenotypic heterogeneity, is the consequence of cell-to-cell differences in gene expression (not linked to genetic differences) among the bacterial community. To analyze the expression of individual loci at the single-cell level, transcriptional fusions to fluorescent proteins have been widely used. Under stress conditions, such as those occurring during colonization of the plant apoplast, P. syringae differentiates into distinct subpopulations based on the heterogeneous expression of key virulence genes (i.e., the Hrp type III secretion system). However, single-cell analysis of any given P. syringae population recovered from plant tissue is challenging due to the cellular debris released during the mechanical disruption intrinsic to the inoculation and bacterial extraction processes. The present report details a method developed to monitor the expression of P. syringae genes of interest at the single-cell level during the colonization of Arabidopsis and bean plants. The preparation of the plants and the bacterial suspensions used for inoculation using a vacuum chamber are described. The recovery of endophytic bacteria from infected leaves by apoplastic fluid extraction is also explained here. Both the bacterial inoculation and bacterial extraction methods are empirically optimized to minimize plant and bacterial cell damage, resulting in bacterial preparations optimal for microscopy and flow cytometry analysis.

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

The present protocol describes a method that allows single-cell gene expression analysis on Pseudomonas syringae populations grown within the plant apoplast.

A plethora of pathogenic microorganisms constantly attack plants. The Pseudomonas syringae species complex encompasses Gram-negative plant-pathogenic ...

An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation

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. (2019)
This video demonstrates an assay to measure oxidative burst in Arabidopsis leaves upon immune elicitation. Leaf discs, cut from plant leaves, are incubated with bacterial peptides functioning as microbe-associated molecular patterns or MAMPs. Upon detection of the MAMPs, plant cells induce the production of extracellular ROS, which is detected using a chemiluminescence assay.

1. Detection of ROS burst in Arabidopsis leaf discs following immune elicitation

	Plant growth and maintenance.
	
		To synchronize germination, stratify ...

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

Summary

Explore More Videos

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

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

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

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

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

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

Single-Cell Analysis of the Expression of Pseudomonas syringae Genes within the Plant Tissue

An Assay to Detect Oxidative Burst in Arabidopsis Leaf Discs upon Immune Elicitation