Name: high-throughput identification of resistance to pseudomonas syringae pv. tomato in tomato using seedling flood assay
Uploaded: 2020-03-10T14:00:00
Description: Watch this Scientific Journal Video about High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay at JoVE.com

We thank Jamie Calma for testing the effect of media volume on disease or resistance outcomes. We thank Dr. Ma&#235;l Baudin and Dr. Karl J. Scheiber from the Lewis Lab for providing constructive comments and suggestions on the manuscript. Research on plant immunity in the Lewis laboratory was supported by the USDA ARS 2030-21000-046-00D and 2030-21000-050-00D (JDL), and the NSF Directorate for Biological Sciences IOS-1557661 (JDL).

1. Preparation and use of biosafety cabinet
<ol>
	<li>Wipe down biosafety cabinet with 70% ethanol.</li>
	<li>Close the sash and turn on the ultraviolet light in the biosafety cabinet for 15 min.</li>
	<li>After 15 min, turn off the ultraviolet light in the biosafety cabinet. Lift the sash and turn on the blower for 15 min.</li>
	<li>Wipe all items to be used in the biosafety cabinet with 70% ethanol prior to putting the items into the sterilized cabinet.</li>
	<li>Clean gloves or bare hands with 70% ethanol before wor...

<ol>
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A protocol for flood inoculation with PstDC3000 or Pst19 optimized to detect resistance to these bacterial strains in tomato seedlings is described. There are several critical parameters for optimal results in the seedling resistance assay, including bacterial concentration and surfactant concentration, which were empirically determined22. For PstDC3000, the optical density was optimized to achieve complete survival on a resistant cultivar containing the Pto/Prf...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/60805">High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay</a>. The Introduction, Protocol, Representative Results and Discussion&#160;sections were&#160;updated.

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

Pseudomonas syringae is a Gram-negative pathogenic bacterium that infects a wide range of plant hosts. Bacteria enter the host plant through the stomata or physical wounds and proliferate in the apoplast1. Plants have evolved a two-tiered immune response to protect against infection by bacterial pathogens. The first level occurs at the plant cell surface, where pattern recognition receptors on the plant cell membrane perceive highly conserved pathogen-associated molecular patterns (PAMPs) in a process called PAMP-triggered immunity (PTI)2. During this process, the host plant upregulates defense response pathways, including deposition of callose to the cell wall, closure of stomata, production of reactive oxygen species, and induction of pathogenesis-related genes.
Bacteria can overcome PTI by utilizing a type III secretion system to deliver proteins, called effectors, directly into the plant cell3. Effector proteins commonly target components of PTI and promote pathogen virulence4. The second tier of plant immunity occurs within the plant cell upon recognition of the effector proteins. This recognition is dependent on resistance genes, which encode nucleotide-binding site leucine-rich repeat containing receptors (NLRs). NLRs are capable of either recognizing effectors directly or recognizing their activity on a virulence target or decoy5. They then trigger a secondary immune response in a process called effector-triggered immunity (ETI), which is often associated with a hypersensitive response (HR), a form of localized cell death at the site of infection6. In contrast to gene-for-gene resistance associated with ETI, plants can exhibit quantitative partial resistance, which is dependent on the contribution of multiple genes7.
P. syringae pv. tomato (Pst) is the causal agent of bacterial speck on tomato and is a persistent agricultural problem. Predominant strains in the field have typically been Pst race 0 strains that express either or both of the type III effectors AvrPto and AvrPtoB. DC3000 (PstDC3000) is a representative race 0 strain and a model pathogen that can cause bacterial speck in tomato. To combat bacterial speck disease, breeders introgressed the Pto [P. syringae pv. tomato]/Prf [Pto resistance and fenthion sensitivity] gene cluster from the wild tomato species Solanum pimpinellifolium into modern cultivars8,9. The Pto gene encodes a serine-threonine protein kinase that, together with the Prf NLR, confer resistance to PstDC3000 via recognition of the effectors AvrPto and AvrPtoB10,11,12,13,14. However, this resistance is ineffective against emerging race 1 strains, allowing for their rapid and aggressive spread in recent years15,16. Race 1 strains evade recognition by the Pto/Prf cluster, because AvrPto is either lost or mutated in these strains, and AvrPtoB appears to accumulate minimally15,17,18.
Wild tomato populations are important reservoirs of natural variation for Pst resistance and have previously been used to identify potential resistance loci19,20,21. However, current screens for pathogen resistance utilize 4&#8211;5-week-old adult plants20,21. Therefore, they are limited by growth time, growth chamber space, and relatively small sample sizes. To address the limitations of conventional approaches, we developed a high-throughput tomato P. syringae resistance assay using 10-day-old tomato seedlings22. This approach offers several advantages over using adult plants: namely, shorter growth time, reduced space requirements, and higher throughput. Furthermore, we have demonstrated that this approach faithfully recapitulates disease resistance phenotypes observed in adult plants22.
In the seedling flood assay described in this protocol, tomato seedlings are grown on Petri dishes of sterile Murashige and Skoog (MS) media for 10 days and then are flooded with an inoculum containing the bacteria of interest and a surfactant. Following flooding, seedlings can be quantitatively evaluated for disease resistance via bacterial growth assays. Additionally, seedling survival or death can act as a discrete resistance or disease phenotype 7&#8211;14 days after flooding. This approach offers a high-throughput alternative for screening large numbers of wild tomato accessions for resistance to Pst race 1 strains, such as Pst strain 19 (Pst19), and can easily be adapted to other bacterial strains of interest.

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			<td>3M Tape Micropore 1/2&#34; x 10 YD CS 240 (1.25 cm x 9.1 m)</td>
			<td>VWR International</td>
			<td>56222-182</td>
			<td></td>
		</tr>
		<tr>
			<td>3mm borosilicate glass beads</td>
			<td>Friedrich &#38; Dimmock</td>
			<td>GB3000B</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto peptone</td>
			<td>BD</td>
			<td>211677</td>
			<td></td>
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		<tr>
			<td>Bacto agar</td>
			<td>BD</td>
			<td>214010</td>
			<td></td>
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			<td>Biophotometer Plus</td>
			<td>Eppendorf</td>
			<td>E952000006</td>
			<td></td>
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			<td>Biosafety cabinet, class II type A2</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>BRAND Disposable Plastic Cuvettes, Polystyrene</td>
			<td>VWR International</td>
			<td>47744-642</td>
			<td></td>
		</tr>
		<tr>
			<td>Chenille Kraft Flat Wood Toothpicks</td>
			<td>VWR International</td>
			<td>500029-808</td>
			<td></td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Research Products International</td>
			<td>C81040-5.0</td>
			<td></td>
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		<tr>
			<td>Dibasic potassium phosphate anhydrous, ACS grade</td>
			<td>Fisher Scientific</td>
			<td>P288-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethylformamide</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope (Magnification of at least 10x)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol - 190 Proof</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon polystyrene 96 well microplates, flat-bottom</td>
			<td>Fisher Scientific</td>
			<td>08-772-3</td>
			<td></td>
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			<td>Glass Alcohol Burner Wick</td>
			<td>Fisher Scientific</td>
			<td>S41898A / No. W-125</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Alcohol Burners</td>
			<td>Fisher Scientific</td>
			<td>S41898 / No. BO125</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol ACS reagent</td>
			<td>VWR International</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
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			<td>Kimberly-Clark&#8482; Kimtech Science&#8482; Kimwipes&#8482; Delicate Task Wipers</td>
			<td>Fisher Scientific</td>
			<td>06-666-A</td>
			<td></td>
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			<td>Magnesium chloride, ACS grade</td>
			<td>VWR International</td>
			<td>97061-356</td>
			<td></td>
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			<td>Magnesium sulfate heptahydrate, ACS grade</td>
			<td>VWR International</td>
			<td>97062-130</td>
			<td></td>
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			<td>Microcentrifuge tubes, 1.5 mL</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Microcentrifuge tubes, 2.2 mL</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Mini Beadbeater-96, 115 volt</td>
			<td>Bio Spec Products Inc.</td>
			<td>1001</td>
			<td></td>
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		<tr>
			<td>Murashige &#38; Skoog, Basal Salts</td>
			<td>Caisson Laboratories, Inc.</td>
			<td>MSP01-50LT</td>
			<td></td>
		</tr>
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			<td>Pipet-Lite XLS LTS 8-CH Pipet 20-200uL</td>
			<td>Rainin</td>
			<td>L8-200XLS</td>
			<td></td>
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			<td>Pipet-Lite XLS LTS 8-CH Pipet 2-20uL</td>
			<td>Rainin</td>
			<td>L8-20XLS</td>
			<td></td>
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			<td>Polystyrene 100mm x 25mm sterile petri dish</td>
			<td>VWR International</td>
			<td>89107-632</td>
			<td></td>
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			<td>Polystyrene 150mm x 15mm sterile petri dish</td>
			<td>Fisher Scientific</td>
			<td>FB08-757-14</td>
			<td></td>
		</tr>
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			<td>Polystyrene 150x15mm sterile petri dish</td>
			<td>Fisher Scientific</td>
			<td>08-757-148</td>
			<td></td>
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			<td>Pure Bright Germicidal Ultra Bleach 5.7% Available Chlorine (defined as 100% bleach)</td>
			<td>Staples</td>
			<td>1013131</td>
			<td></td>
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			<td>Rifampicin</td>
			<td>Gold Biotechnology</td>
			<td>R-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Silwet L-77 (non-ionic organosilicone surfactant co-polymer C13H34O4Si3 surfactant)</td>
			<td>Fisher Scientific</td>
			<td>NCO138454</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips LTS 20 &#956;L 960/10 GPS-L10</td>
			<td>Rainin</td>
			<td>17005091</td>
			<td></td>
		</tr>
		<tr>
			<td>Tips LTS 250 &#956;L 960/10 GPS-L250</td>
			<td>Rainin</td>
			<td>17005093</td>
			<td></td>
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		<tr>
			<td>VWR dissecting forceps fine tip, 4.5&#34;</td>
			<td>VWR International</td>
			<td>82027-386</td>
			<td></td>
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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.

Detection of PtoR-mediated immunity in cultivars and isogenic lines using the seedling resistance assay 
Figure 5 shows representative results for Moneymaker-PtoR and Moneymaker-PtoS cultivars 7&#8211;10 days after flooding with PstDC3000. Prior to infection, 10-day-old seedlings displayed fully emerged and expanded cotyledons and emerging first true leaves. The seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as...

Watch this Scientific Journal Video about High-Throughput Identification of Resistance to Pseudomonas syringae pv. Tomato in Tomato using Seedling Flood Assay at JoVE.com

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.

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. ...

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Bioengineering

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