Name: assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants
Uploaded: 2009-09-09T16:30:00
Duration: 8 min 45 s
Description: Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

We thank Hye-Sook Oh, Joanne Morello and Alan Collmer, Department of Plant Pathology and Plant-Microbe Biology, Cornell University, for valuable discussions. We thank Jesse Munkvold for comments on the article. Funding was provided by the National Science Foundation Plant Genome Program, award number DBI-0605059 (GBM).

Part 1: Plant growth and maintenance
Nicotiana benthamiana plants used in the assay should be about 7 weeks old. They should be trimmed at least 4-5 days prior to the assay to remove all axillary branches and flowers. It is a good idea to remove axillary branches soon after they emerge in order to make the plants more manageable.
Part 2: Bacterial culture
DAY 1:
<ol>
<li>Plates or cultures for all the strains used in the assay are initiated at the same time. For the Pseudomonas spp. which include 2 inducers and 3 challenge strains, streak bacteria onto KBM plates containing the appropriate antibiotics from frozen glycerol stocks (Table 2). A small amount of bacteria should be spread on the center of the plate. Incubate the plate at 30&deg;C for about 18-24 hours. For Agrobacterium, start a liquid culture in 2 ml LB from a fresh plate and grow overnight at 250 r.p.m., 30&deg;C.</li>
</ol>
DAY 2:
<ol start="2">
<li>For Pseudomonas, add 150-200 &mu;l of liquid KBM to the bacteria and spread them over the entire plate using a sterile glass spreader. Put the plate back in the incubator and let it grow for another 20-24 hours. There should be a bacterial lawn on the plate the next day, indicating good growth. For Agrobacterium, start a secondary culture in about 5-10 ml LB with 20 &mu;M acetosyringone and let it grow overnight to 20 hours at 250 r.p.m., 30&deg;C.</li>
</ol>
Part 3: Preparing plants for the assay
DAY 2:
<ol>
<li>One day before the assay the plants should be moved to a room at 22-24&deg;C, ~35-40% RH and constant light.</li>
<li>Mark circles on leaves that are at least 1.5 cm in diameter using a thick black marker. Two to four circles can be marked per leaf. The circles should be well spaced and preferably not cross a large vein. Choose well-expanded leaves. Avoid older leaves or leaves that are tough or thick to the touch and avoid marking circles on the lower portions of the leaf.</li>
</ol>
Marking circles a day prior to the experiment is not essential to the protocol. However, it saves time if there are large numbers of plants being assayed, and makes it easier to achieve precise timing between the induction of PTI and challenge inoculations.
Part 4: Induction of PTI
DAY 3:
<ol>
<li>Harvest the cells from the plate in 10 mM MgCl2 for P. fluorescens and P. putida. For Agrobacterium, centrifuge the culture to collect cells and resuspend in 10mM MgCl2 + 10 mM MES pH 5.6. Repeat the centrifugation and resuspend in the MgCl2 -MES solution. Measure the optical density (O.D.) of the cells at 600 nM. Adjust the O.D.s to the final required values as described in Table 2. Pseudomonas should be finally resuspended in 10 mM MgCl2 and Agrobacterium in the MgCl2 &#x2013;MES solution. A total volume of 25 ml is sufficient to inoculate about 40-50 spots.</li>
<li>Infiltrate the PTI inducers into the pre-marked circles on the leaves using a 1 ml needleless syringe. Write down the order in which the plants were infiltrated and the exact time when the infiltration was begun.</li>
</ol>
Part 5: Challenge inoculation
<ol>
<li>Prepare P. syringae pv. tomato DC3000, P.s.t. DC3000 &Delta;hopQ1-1 and P.s. pv. tabaci for the challenge as described in part 4.1 and Table 2.</li>
<li>Seven hours after the inducer infiltration, perform the challenge infiltration in the same order of plants as the induction was done. Generally, a point on the periphery of the first inoculation circle can be used as the center of the second inoculation circle. If there are multiple spots on a leaf then make sure the infiltrations do not overlap.</li>
<li>Plate serial dilutions of all the cultures used in the assay on KBM or LB plates containing the appropriate antibiotics to determine the exact C.F.U. number.</li>
</ol>
Part 6: Scoring for breakdown of PTI
Day 5:
<ol>
<li>Look for the appearance of cell death due to ETI in the spots that were challenged with P.s.t. DC3000. Cell death inside the overlapping area of infiltration indicates a breakdown of PTI and should be scored as a positive phenotype.</li>
</ol>
Day 7:
<ol start="2">
<li>Look for the appearance of cell death due to disease in the spots that were challenged with P.s.t. DC3000 &Delta;hopQ1-1 or P.s. pv. tabaci. Score for a positive phenotype in the same manner as described in part 6.1.</li>
</ol>
When the control plants (that should not be compromised for PTI) begin to show cell death in the overlapping area of infiltration, stop making any further observations.
Part 7: Representative results
Figure 1 shows the outcome of an assay when P. fluorescens was used as the inducer and P.s.t. DC3000 as the challenge.
<img src="/files/ftp_upload/1442/fig1tmb.jpg" alt="Figure 1" border="0" /> Figure 1. P. fluorescens was infiltrated onto N. benthamiana leaves (black circle) to induce PTI and 7 hours later, the spot was challenged with P.s.t DC3000 (white circle). Plants silenced for FLS2 showed a breakdown of PTI in the region where P. fluorescens was infiltrated (A), as seen by cell death. Control plants that were not silenced showed no cell death in the overlapping area due to induction of PTI (B). Red arrows indicate lack of or presence of cell death in the overlapping area of infiltration. Photographs were taken 2 days after the infiltrations. Please <a href="https://www.jove.com/files/ftp_upload/1442/fig1.jpg">click here</a> to see a larger version of figure 1.
<table border="1">
<tbody>
<tr>
<td>PTI inducer</td>
<td>Cell death-eliciting challenge </td>
<td>Nature of cell death </td>
<td>Code </td>
</tr>
<tr>
<td>Pseudomonas fluorescens 55</td>
<td>Pseudomonas syringae pv tomato (P.s.t.) DC30006</td>
<td>ETI</td>
<td>Pf/DC</td>
</tr>
<tr>
<td>P. putida KT2440</td>
<td>P.s.t. DC3000</td>
<td>ETI</td>
<td>Pp/DC</td>
</tr>
<tr>
<td>P. fluorescens 55</td>
<td>P.s.t. DC3000 &Delta;hopQ1-16</td>
<td>Disease</td>
<td>Pf/Q1-1</td>
</tr>
<tr>
<td>Agrobacterium tumefaciens GV2260</td>
<td>P. syringae pv. tabaci 11528R</td>
<td>Disease</td>
<td>Agro/Ptab</td>
</tr>
</tbody>
</table>
Table 1: Combinations of PTI inducing and cell death-eliciting microbes used in the cell death assay for PTI.
<table border="1">
<tbody>
<tr>
<td>Bacterial strain</td>
<td>Selection medium</td>
<td>Final O.D. used in experiment</td>
<td>Corresponding C.F.U./ml </td>
</tr>
<tr>
<td>P. fluorescens 55</td>
<td>KBM Ampicillin (100 ug/ml)</td>
<td>0.5</td>
<td>1 x 109</td>
</tr>
<tr>
<td>P. putida KT2440</td>
<td>KBM Ampicillin (100 ug/ml)</td>
<td>0.5</td>
<td>1 x 108</td>
</tr>
<tr>
<td>A. tumefaciens GV2260</td>
<td>LB Rifampicin (100 ug/ml)</td>
<td>0.5</td>
<td>5 x 108</td>
</tr>
<tr>
<td>P.s.t. DC3000</td>
<td>KBM Rifampicin (100 ug/ml)</td>
<td>0.02</td>
<td>2 x 107</td>
</tr>
<tr>
<td>P.s.t. DC3000 &Delta;hopQ1-1</td>
<td>KBM Rifampicin (100 ug/ml)</td>
<td>100 fold dilution of 0.1</td>
<td>1 x 106</td>
</tr>
<tr>
<td>P. syringae pv. tabaci 11528R</td>
<td>KBM Rifampicin (100 ug/ml)</td>
<td>100 fold dilution of 0.1</td>
<td>1 x 106</td>
</tr>
</tbody>
</table>
Table 2:Culture conditions and inoculum levels used in the assay. The O.D. and corresponding C.F.U. levels may vary between different brand of spectrophotometers.

<ol>
	<li>Abramovitch, R. B., Anderson, J. C., Martin, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+elicitation+and+evasion+of+plant+innate+immunity.">Bacterial elicitation and evasion of plant innate immunity.</a> Nat Rev Mol Cell Biol. 7 (8), 601-60 (2006).</li><li>Jones, J. D., Dangl, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+plant+immune+system.">The plant immune system.</a> Nature. 444 (7117), 323-323 (2006).</li><li>Zipfel, C., Robatzek, S., Navarro, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception.">Bacterial disease resistance in Arabidopsis through flagellin perception.</a> Nature. 428 (6984), 764-764 (2004).</li><li>Oh, H. S., Collmer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+resistance+against+bacteria+in+Nicotiana+benthamiana+leaves+is+accompanied+by+reduced+vascular+staining+and+suppressed+by+multiple+Pseudomonas+syringae+type+III+secretion+system+effector+proteins.">Basal resistance against bacteria in Nicotiana benthamiana leaves is accompanied by reduced vascular staining and suppressed by multiple Pseudomonas syringae type III secretion system effector proteins.</a> Plant J. 44 (2), 348-348 (2005).</li><li>Goodin, M. M., Zaitlin, D., Naidu, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nicotiana+benthamiana:+its+history+and+future+as+a+model+for+plant-pathogen+interactions.">Nicotiana benthamiana: its history and future as a model for plant-pathogen interactions.</a> Mol Plant Microbe Interact. 21 (8), 1015-1015 (2008).</li><li>Wei, C. F., Kvitko, B. H., Shimizu, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Pseudomonas+syringae+pv.+tomato+DC3000+mutant+lacking+the+type+III+effector+HopQ1-1+is+able+to+cause+disease+in+the+model+plant+Nicotiana+benthamiana.">A Pseudomonas syringae pv. tomato DC3000 mutant lacking the type III effector HopQ1-1 is able to cause disease in the model plant Nicotiana benthamiana.</a> Plant J. 51 (1), 32-32 (2007).</li></ol>

The cell death-based assay can be used to determine the involvement of a gene in PTI. For instance, loss-of-function mutants for a gene of interest can be tested using this assay. Out of the 4 combinations of inducer and challenge inoculations given in Table 1, it is possible that only one combination may result in a phenotype in your plant background. We have observed that plants silenced for FLS2 show a breakdown of PTI in the Pf/DC and Pf/Q1-1 combinations of inducer and challenge inoculations (Table 1).
It is essential that the environmental conditions for the assay are as close as possible to those described in Part 3.1. Low humidity causes cell death to proceed too quickly, making the assay difficult to score. If the conditions described in our protocol do not work in your lab, then try altering the level of inducer or challenge inoculation. Another parameter that can be modified is the time gap between induction and challenge, although we have found that shorter time gaps caused the assay to break down too quickly in control plants.
We recommend that at least 4-5 plants be tested in an experiment and a positive phenotype should be repeated in at least 3-4 independent experiments.

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<td>MES</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP300-100</td>
<td>Prepare as a1M stock, adjust pH and filter sterilize. Store at room temperature. </td>
</tr>
<tr>
<td>Life Science UV/Vis Spectrophotometer</td>
<td>&nbsp;</td>
<td>Beckman Coulter</td>
<td>DU 730</td>
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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.

Watch this Scientific Journal Video about Assay for Pathogen-Associated Molecular Pattern PAMP-Triggered Immunity PTI in Plants at JoVE.com

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.

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 ...

assay-for-pathogen-associated-molecular-pattern-pamp-triggered

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Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in humans. Several mouse models of gastric Helicobacter infection have been developed to study the molecular and cellular mechanisms whereby H. pylori bacteria colonize the stomach of human hosts and cause disease. Herein, we describe protocols to: 1) prepare bacterial suspensions for the in vivo infection of mice via intragastric gavage; 2) determine bacterial colonization levels in mouse gastric tissues, by polymerase chain reaction (PCR) and viable counting; and 3) assess pathological changes, by histology. To establish Helicobacter infection in mice, specific pathogen-free (SPF) animals are first inoculated with suspensions (containing &#8805;105 colony-forming units, CFUs) of mouse-colonizing strains of either Helicobacter pylori or other gastric Helicobacter spp. from animals, such as Helicobacter felis. At the appropriate time-points post-infection, stomachs are excised and dissected sagittally into two equal tissue fragments, each comprising the antrum and body regions. One of these fragments is then used for either viable counting or DNA extraction, while the other is subjected to histological processing. Bacterial colonization and histopathological changes in the stomach may be assessed routinely in gastric tissue sections stained with Warthin-Starry, Giemsa or Haematoxylin and Eosin (H&#38;E) stains, as appropriate. Additional immunological analyses may also be undertaken by immunohistochemistry or immunofluorescence on mouse gastric tissue sections. The protocols described below are specifically designed to enable the assessment in mice of gastric pathologies resembling those in human-related H. pylori diseases, including inflammation, gland atrophy and lymphoid follicle formation. The inoculum preparation and intragastric gavage protocols may also be adapted to study the pathogenesis of other enteric human pathogens that colonize mice, such as Salmonella Typhimurium or Citrobacter rodentium.

Mouse Models Of Helicobacter Infection And Gastric Pathologies

Mice represent an invaluable in vivo model to study infection and diseases caused by gastrointestinal microorganisms. Here, we describe the methods used to study bacterial colonization and histopathological changes in mouse models of Helicobacter pylori-related disease.

Helicobacter pylori is a gastric pathogen that is present in half of the global population and is a significant cause of morbidity and mortality in ...

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our knowledge of prevention and treatment of viral infection. The fruit fly Drosophila melanogaster has proven to be one of the most efficient and productive model organisms to screen for antiviral factors and investigate virus-host interaction, due to powerful genetic tools and highly conserved innate immune signaling pathways. The procedure described here demonstrates a nano-injection method to establish viral infection and induce systemic antiviral responses in adult flies. The precise control of the viral injection dose in this method enables high experimental reproducibility. Protocols described in this study include the preparation of flies and the virus, the injection method, survival rate analysis, the virus load measurement, and an antiviral pathway assessment. The influence effects of viral infection by the flies' background were mentioned here. This infection method is easy to perform and quantitatively repeatable; it can be applied to screen for host/viral factors involved in virus-host interaction and to dissect the crosstalk between innate immune signaling and other biological pathways in response to viral infection.

Establishment of Viral Infection and Analysis of Host-Virus Interaction in Drosophila Melanogaster

This protocol describes how to establish viral infection in vivo in Drosophila melanogaster using the nano-injection method and basic techniques to analyze virus-host interaction.

Virus spreading is a major cause of epidemic diseases. Thus, understanding the interaction between the virus and the host is very important to extend our ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...