Name: high-throughput identification of resistance to pseudomonas syringae pv. tomato in tomato using seedling flood assay
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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).

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The authors have nothing to disclose.

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 cluster and complete death on a susceptible cultivar lacking the Pto/Prf cluster22. For a strain such as Pst19, where there are no known resistant varieties, the optical density was optimized to be the lowest possible for consistent and complete plant death22. Uppalapati et al.24 designed a tomato seedling assay to investigate the pathogenesis of PstDC3000 and the virulence function of coronatine. In this virulence assay, infections were performed using bacteria concentrated to an OD600 of 0.124, 20x higher than the optical density of strains used in our resistance assay. Recognition of PstDC3000 effectors AvrPto and AvrPtoB in tomato seedlings carrying the Pto/Prf gene cluster results in ETI and a macroscopic HR22. In the context of a strong immune response such as ETI, a lower bacterial titer was used for PstDC3000 to avoid overwhelming genetic resistance from the Pto/Prf gene cluster22. In addition, these results suggest that a high bacterial concentration could overwhelm weaker immune responses such as PTI or quantitative partial resistance, where multiple genes contribute to the overall phenotype. Surfactant is necessary for the bacteria to adhere to the leaf surface; however, high concentrations can cause chlorosis of the leaf22. We previously tested a range of surfactant concentrations to empirically determine the ideal concentration in 10-day-old tomato seedlings22. When testing new species that may differ in their sensitivity to surfactant, the surfactant concentration should be optimized to identify a concentration that does not cause damage or chlorosis in the absence of bacteria. Appropriate assay conditions will require optimization of a surfactant concentration that does not cause damage, and a bacterial concentration that causes disease in all susceptible controls.
Additional critical parameters for success in the seedling flood assay include using seedlings at specific developmental stages (10-day-old seedlings) (Figure 1), maintaining stable growth chamber conditions (light intensity of about 200 &#181;E m-2 s-1, constant temperature of 22 &#730;C, 16 h of light) and performing experiments in a sterile biosafety cabinet. Media volume above 45 mL or below 35 mL may affect consistent death of susceptible controls, because the volume may impact the surrounding microenvironment of the seedlings on the plate. For example, differences in relative humidity inside the sealed plates could affect the infectivity of the bacteria and the ability of the plants to survive infection. Sterile technique is critical, because contamination on the plates may confound the source of death or susceptibility in seedlings. In addition, because plant-pathogen interactions are affected by the circadian clock24,25,26, it is recommended that the plants be infected at a consistent time of the day.
Pst is a foliar pathogen that preferentially colonizes the aerial parts of tomato seedlings, including the cotyledons24 (Figure 3). Therefore, qualitative phenotyping in the seedling flood assay focuses on growth and disease symptoms in aerial portions of the seedling, and tissue for the bacterial growth assay is sampled from the cotyledons for quantitative analysis. After flood inoculation, seedlings may die within 7&#8211;10 days after inoculation with PstDC3000 or 10&#8211;14 days after inoculation with Pst19, as discussed in section 11. Seedling death is visualized by a brown apical meristem, arrested epicotyl elongation, and/or arrested vegetative growth. If different bacterial strains are used, the timing will have to be empirically determined. In addition, the progression of disease on control plants should be monitored daily after flooding until a consistent time frame from the onset of disease symptoms to seedling death can be identified. Depending on the genotypes and treatments used in the flood assay, seedling phenotypes can be recorded as binary phenotypes or on a disease spectrum (Figure 4). A broader spectrum of phenotypes may be observed when flood inoculating F2 mapping populations from wild tomato accessions crossed to susceptible cultivars (Figure 4C). It may be best to phenotype segregating populations on a disease spectrum depending on how quickly the seedling dies and the degree of new vegetative growth and branching (Figure 4C). The seedling flood assay can also be used in conjunction with the seedling bacterial growth assay to quantitatively assess levels of bacterial growth associated with qualitative phenotypes in individual seedlings (Figure 7). Very large reductions (i.e., ~log 3) in bacterial growth or strong resistance in resistant seedlings of a wild accession compared to a susceptible cultivar suggest that the underlying genetic basis of resistance may be due to ETI22. Smaller reductions in bacterial growth (i.e., ~log 1.7), as observed in LA1329 seedlings, may be due to the contribution of weaker resistance from quantitative trait loci and/or PTI. Thus, the seedling growth assay can be an important tool in further characterizing resistance in wild tomato lines.
Typically, genetic screens have been performed on four- to five-week-old adult tomato plants to identify the genetic basis of P. syringae resistance in wild accessions20,21. Adult tomato plants require much longer growth times, require more space in the growth chamber, and are much larger plants, which means that usually few individuals are screened for each line. The seedling flood assay provides a powerful, alternative approach in the identification of P. syringae resistance in wild tomato accessions. Screening at the seedling stage permits a large sample size to be tested which can be particularly advantageous in detecting resistance in genetically complex populations. Reduced growth chamber space requirements and growth time facilitate a high-throughput approach and rapid detection of natural resistance in wild accessions to emerging pathogens. Furthermore, P. syringae resistance that was identified at the seedling stage in this assay is not restricted to the developmental stage. S. neorickii LA1329 and S. habrochaites LA1253 were initially identified at the seedling stage and also display resistance to Pst19 in adult plants as previously described22.
The seedling flood assay is a versatile protocol that can be modified and optimized to detect host resistance to other P. syringae strains. It could potentially be further applied in the context of different bacterial pathogens of tomato, such as the Xanthomonas species. This method will expedite the search for new sources of disease resistance to bacterial pathogens.

An erratum was issued for:&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/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.

<table><tbody><tr><td>3M Tape Micropore 1/2&quot; 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 &amp;amp; Dimmock</td><td>GB3000B</td><td></td></tr><tr><td>Bacto peptone</td><td>BD</td><td>211677</td><td></td></tr><tr><td>Bacto agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Biophotometer Plus</td><td>Eppendorf</td><td>E952000006</td><td></td></tr><tr><td>Biosafety cabinet, class II type A2</td><td></td><td></td><td></td></tr><tr><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></tr><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></tr><tr><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><tr><td>Kimberly-Clark&amp;trade; Kimtech Science&amp;trade; Kimwipes&amp;trade; Delicate Task Wipers</td><td>Fisher Scientific</td><td>06-666-A</td><td></td></tr><tr><td>Magnesium chloride, ACS grade</td><td>VWR International</td><td>97061-356</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate, ACS grade</td><td>VWR International</td><td>97062-130</td><td></td></tr><tr><td>Microcentrifuge tubes, 1.5 mL</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge tubes, 2.2 mL</td><td></td><td></td><td></td></tr><tr><td>Mini Beadbeater-96, 115 volt</td><td>Bio Spec Products Inc.</td><td>1001</td><td></td></tr><tr><td>Murashige &amp;amp; Skoog, Basal Salts</td><td>Caisson Laboratories, Inc.</td><td>MSP01-50LT</td><td></td></tr><tr><td>Pipet-Lite XLS LTS 8-CH Pipet 20-200uL</td><td>Rainin</td><td>L8-200XLS</td><td></td></tr><tr><td>Pipet-Lite XLS LTS 8-CH Pipet 2-20uL</td><td>Rainin</td><td>L8-20XLS</td><td></td></tr><tr><td>Polystyrene 100mm x 25mm sterile petri dish</td><td>VWR International</td><td>89107-632</td><td></td></tr><tr><td>Polystyrene 150mm x 15mm sterile petri dish</td><td>Fisher Scientific</td><td>FB08-757-14</td><td></td></tr><tr><td>Polystyrene 150x15mm sterile petri dish</td><td>Fisher Scientific</td><td>08-757-148</td><td></td></tr><tr><td>Pure Bright Germicidal Ultra Bleach 5.7% Available Chlorine (defined as 100% bleach)</td><td>Staples</td><td>1013131</td><td></td></tr><tr><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 C&lt;sub&gt;13&lt;/sub&gt;H&lt;sub&gt;34&lt;/sub&gt;O&lt;sub&gt;4&lt;/sub&gt;Si&lt;sub&gt;3&lt;/sub&gt; surfactant)</td><td>Fisher Scientific</td><td>NCO138454</td><td></td></tr><tr><td>Tips LTS 20 &amp;mu;L 960/10 GPS-L10</td><td>Rainin</td><td>17005091</td><td></td></tr><tr><td>Tips LTS 250 &amp;mu;L 960/10 GPS-L250</td><td>Rainin</td><td>17005093</td><td></td></tr><tr><td>VWR dissecting forceps fine tip, 4.5&quot;</td><td>VWR International</td><td>82027-386</td><td></td></tr></tbody></table>

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 a negative control (data not shown) and PstDC3000 at an optical density of 0.005 + 0.015% surfactant. The seedlings were phenotyped 7&#8211;10 days after flooding (Figure 5). Individual seedlings from genotypically homogenous lines, such as Moneymaker-PtoR and Moneymaker-PtoS give highly consistent and binary phenotypes in the seedling flood assay. When Moneymaker-PtoR, which carries the Pto/Prf gene cluster (n = 5), was treated with PstDC3000 at the optimal concentration of OD600 = 0.005, resistance due to PtoR-mediated immunity was strong and was typified by new, green vegetative growth in all individuals22. Near-isogenic Moneymaker-PtoS seedlings (n = 5), which cannot recognize the PstDC3000 effectors AvrPto or AvrPtoB, died quickly within 7 days after flooding and characteristically had brown apical meristems, bacterial speck, chlorosis, and no signs of new, green vegetative growth (Figure 5).
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60805/60805fig05.jpg" /> 
Figure 5: Phenotypic characterization of resistance or disease symptoms 7&#8211;10 days post-infection in a cultivar. Moneymaker-PtoR and Moneymaker-PtoS tomato seedlings were grown on 0.5x MS plates for 10 days before being flooded with P. syringae pv. Tomato DC3000 (OD600 = 0.005) + 0.015% surfactant. Moneymaker-PtoR seedlings survived (n = 5) and Moneymaker-PtoS seedlings (n = 5) died. The number of surviving seedlings for each genotype out of the total number tested is shown. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60805/60805fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Phenotypic screening of wild accessions using the seedling resistance assay 
Figure 6 shows representative results for seedlings of susceptible and resistant accessions 10&#8211;14 days after flooding with Pst19. Susceptible accessions include RG-PtoR, S. pimpinellifolium LA1375, and S. pimpinellifolium LA1606, and resistant accessions include S. neorickii LA1329. Ten-day-old seedlings were flooded with 10 mM MgCl2 + 0.015% surfactant as a negative control, and Pst19 at an optical density of 0.0075 + 0.015% surfactant. The seedlings were phenotyped at least 10 days after flooding, as Pst19-infected seedlings died more slowly than PstDC3000-infected seedlings. Mock-inoculated seedlings were green, healthy, and actively growing. This control is important to ensure that the accessions are not sensitive to the concentration of surfactant, and to ensure there is no bacterial contamination. Susceptible accessions (Rio Grande-PtoR [n = 7], S. pimpinellifolium LA1375 [n = 7], and S. pimpinellifolium LA1606 [n = 5]) were dead, had brown apical meristems, and lacked new growth 10&#8211;14 days after inoculation with Pst19. In contrast, two S. neorickii LA1329 (n = 3) seedlings displayed a high level of new, green growth and survived infection with Pst19 (Figure 6). Three LA1329 seedlings did not germinate. Typically, 5&#8211;7 individuals were screened for each accession in a primary screen to determine the prevalence of resistance in the population. When a more genetically complex wild accession, such as LA1329, is flooded with Pst19, the resistance phenotypes display slightly more variability among individual seedlings, compared to Moneymaker-PtoR treated with PstDC3000. However, the resistance phenotypes were usually less variable than those seen in F2 mapping populations. Thus, binary phenotyping criteria was used for LA1329.
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60805/60805fig6v2.jpg" /> 
Figure 6: Phenotypic characterization of resistance or disease symptoms 10&#8211;14 days post-infection in wild accessions. Rio Grande-PtoR, S. pimpinellifolium LA1606, S. pimpinellifolium LA1375 and S. neorickii LA1329 tomato seedlings were grown on 0.5x MS plates for 10 days, and then flooded with Pst19 (OD600 = 0.0075) + 0.015% surfactant. The number of surviving seedlings for each wild accession out of the total number tested is shown. Scale bar = 1 cm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60805/60805fig6v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Quantitative assessment of bacterial growth using the seedling flood assay 
To confirm that the observed resistance in LA1329 to Pst19 resulted in lower bacterial growth, bacterial growth assays were carried out in tomato seedlings. The level of Pst19 growth in Moneymaker-PtoS and S. neorickii LA1329 was determined 4 days post-infection. Moneymaker-PtoS is a near-isogenic line with consistent susceptibility among individual seedlings. Wild accessions such as S. neorickii LA1329 are often more genetically complex. LA1329 displays approximately 60% resistance to Pst19 across the population22. Because seedlings may drop their cotyledons after infection, one seedling was grown on each plate to correlate bacterial growth in the harvested cotyledon with overall seedling survival or death as determined phenotypically at least 10 days after flooding. The bacterial counts on day 4 for each seedling were normalized to 0.01 g of tissue and converted to log growth (CFU/0.01 g(log10)). Log growth for phenotypically resistant LA1329 seedlings (LA1329RES) or phenotypically susceptible seedlings (LA1329SUS) were separately pooled and compared to each other and the susceptible cultivar Moneymaker-PtoS. For example, there was a 1.7 log difference in bacterial growth between LA1329RES (log 6.3) and LA1329SUS (log 8.0), and a 1.6 log difference between LA1329RES (log 6.3) and Moneymaker-PtoS (log 7.9) (Figure 7). Therefore, phenotypic resistance correlated with quantitative resistance in the seedling assays.
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/60805/60805fig7v2.jpg" /> 
Figure 7: Resistant Solanum neorickii LA1329 seedlings support lower bacterial growth than Moneymaker-PtoS or susceptible S. neorickii LA1329. Bacterial counts were determined 4 days post-inoculation from S. neorickii LA1329 (n = 14) and Moneymaker-PtoS (n = 10) seedlings infected with Pst19 and normalization was performed to 0.1 g of tissue. For LA1329, the two phenotypic groups, susceptible (SUS) or resistant (RES), were observed and counted separately. Above the bar * = statistically significant difference determined by a one-factor analysis of variance. A general linear model procedure (p &#60; 0.001) followed by a multiple comparison of means using Tukey&#39;s post hoc test was used. Error bars = standard error. The figure indicates one representative experiment. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60805/60805fig7v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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. ...

high-throughput-identification-resistance-to-pseudomonas-syringae-pv

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.5K Views.  University of California, Berkeley. Dit hoge doorvoerprotocol maakt een snelle screening van tomatenzaailingen mogelijk van wilde toetredingen tot de bacteriële ziekteverwekker Pseudomonas syringae. De zaailing overstromingstest minimaliseert de groeitijd van planten en de groei kamer behoeften, maakt een snelle omzet van planten, en maakt het mogelijk grote steekproef maten worden getest. Deze test is een krachtig instrument dat kan worden gebruikt om te screenen op weerstand in wilde toetredingen en andere lijnen met complex...

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

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 ...

Immunology and Infection

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 ...

Cell-based Calcium Assay for Medium to High Throughput Screening of TRP Channel Functions using FlexStation 3

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates. It is ideal for medium- to high-throughput screens in academic settings. It has an integrated fluid transfer module equipped with a multi-channel pipetter and the machine reads one column at a time to monitor fluorescence changes of a variety of fluorescent reagents. For example, FlexStation 3 has been used to study the function of Ca2+-permeable ion channels and G-protein coupled receptors by measuring the changes of intracellular free Ca2+ levels. Transient receptor potential (TRP) channels are a large family of nonselective cation channels that play important roles in many physiological and pathophysiological functions. Most of the TRP channels are calcium permeable and induce calcium influx upon activation. In this video, we demonstrate the application of FlexStation 3 to study the pharmacological profile of the TRPA1 channel, a molecular sensor for numerous noxious stimuli. HEK293 cells transiently or stably expressing human TRPA1 channels, grown in 96-well plates, are loaded with a Ca2+-sensitive fluorescent dye, Fluo-4, and real-time fluorescence changes in these cells are measured before and during the application of a TRPA1 agonist using the FLEX mode of the FlexStation 3. The effect of a putative TRPA1 antagonist was also examined. Data are transferred from the SoftMax Pro software to construct concentration-response relationships of TRPA1 activators and inhibitors.

This video provides a detailed protocol for studying the pharmacological profile of human TRPA1 channels using FlexStation 3. The protocol covers details of cell preparation, dye loading and operation of the microplate reader, FlexStation 3.

The Molecular Devices' FlexStation 3 is a benchtop multi-mode microplate reader capable of automated fluorescence measurement in multi-well plates.  It is ...

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to agriculture. However, the Ralstonia model is considerably underexplored in comparison to other models involving bacterial plant pathogens, such as Pseudomonas syringae in Arabidopsis. Research targeted to understanding the interaction between Ralstonia and crop plants is essential to develop sustainable solutions to fight against bacterial wilt disease but is currently hindered by the lack of straightforward experimental assays to characterize the different components of the interaction in native host plants. In this scenario, we have developed a method to perform genetic analysis of Ralstonia infection of tomato, a natural host of Ralstonia. This method is based on Agrobacterium rhizogenes-mediated transformation of tomato roots, followed by Ralstonia soil-drenching inoculation of the resulting plants, containing transformed roots expressing the construct of interest. The versatility of the root transformation assay allows performing either gene overexpression or gene silencing mediated by RNAi. As a proof of concept, we used this method to show that RNAi-mediated silencing of SlCESA6 in tomato roots conferred resistance to Ralstonia. Here, we describe this method in detail, enabling genetic approaches to understand bacterial wilt disease in a relatively short time and with small requirements of equipment and plant growth space.

Tomato Root Transformation Followed by Inoculation with Ralstonia Solanacearum for Straightforward Genetic Analysis of Bacterial Wilt Disease

Here, we present a versatile method for tomato root transformation followed by inoculation with Ralstonia solanacearum to perform straightforward genetic analysis for the study of bacterial wilt disease.

Ralstonia solanacearum is a devastating soil borne vascular pathogen that can infect a large range of plant species, causing an important threat to ...

Genetics

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 ...

An Efficient Clearing Protocol for the Study of Seed Development in Tomato (Solanum lycopersicum L.)

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. Visualization of finer embryonic structure within a tomato seed is often hampered by seed coat mucilage, multi-cell-layered integument, and a thick-walled endosperm, which needs to be resolved by laborious embedding-sectioning. A simpler alternative is to employ tissue clearing techniques that turn the seed almost transparent using chemical agents. Although conventional clearing procedures allow deep insight into smaller seeds with a thinner seed coat, clearing tomato seeds continues to be technically challenging, especially in the late developmental stages.
Presented here is a rapid and labor-saving clearing protocol to observe tomato seed development from 3 to 23 days after flowering when embryonic morphology is nearly complete. This method combines chloral hydrate-based clearing solution widely used in Arabidopsis with other modifications, including the omission of Formalin-Aceto-Alcohol (FAA) fixation, the addition of sodium hypochlorite treatment of seeds, removal of the softened seed coat mucilage, and washing and vacuum treatment. This method can be applied for efficient clearing of tomato seeds at different developmental stages and is useful in full monitoring of the developmental process of mutant seeds with good spatial resolution. This clearing protocol may also be applied to deep imaging of other commercially important species in the Solanaceae.

An Efficient Clearing Protocol for the Study of Seed Development in Tomato Solanum lycopersicum L.

The tomato seed is an important model for studying genetics and developmental biology during plant reproduction. This protocol is useful for clearing tomato seeds at different developmental stages to observe the finer embryonic structure.

Tomato (Solanum lycopersicum L.) is one of the major cash crops worldwide. The tomato seed is an important model for studying genetics and developmental ...

Developmental Biology

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 ...

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. P. nicotianae is well adapted to high temperatures; therefore, research on this pathogen is gaining importance in agriculture worldwide because of global warming. P. nicotianae-resistant varieties of tobacco plants are commonly screened by inoculation with oat grains colonized by P. nicotianae and monitoring for the disease symptoms. However, it is difficult to quantify the inoculation intensity since accurate inoculation is crucial in this case. This study aimed to develop an efficient and reliable method for evaluating the resistance of tobacco to infection with P. nicotianae. This method has been successfully used to identify resistant varieties, and the inoculation efficiency was confirmed by real-time PCR. The resistance evaluation method presented in this study is efficient and practical for precision breeding, as well as molecular mechanism research.

Screening of Tobacco Genotypes for Phytophthora nicotianae Resistance

Here, a protocol is presented for the efficient and accurate screening of tobacco genotypes for Phytophthora nicotianae resistance in seedlings. This is a practical approach for precision breeding, as well as molecular mechanism research.

Black shank, caused by the oomycetes Phytophthora nicotianae, is destructive to tobacco, and this pathogen is highly pathogenic to many solanaceous crops. ...

Biology

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 ...

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