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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		<div>
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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

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 ...

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27.0K Views.  Boyce Thompson Institute for Plant Research. A cell death-based assay for PTI in Nicotiana benthamiana plants is described.

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

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

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The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
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Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

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Leucocyte recruitment to the liver occurs within the specialized channels of the hepatic sinusoids which are lined by unique hepatic sinusoidal endothelial cells. Phase contrast microscopy of leucocyte recruitment across human hepatic sinusoidal endothelium under conditions of physiological shear stress can facilitate the elucidation of the molecular mechanisms which underlie this process.

Leucocyte infiltration into human liver tissue is a common process in all adult inflammatory liver diseases. Chronic infiltration can drive the ...

High Yield Purification of Plasmodium falciparum Merozoites For Use in Opsonizing Antibody Assays

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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 ...

Intravital Imaging of Intraepithelial Lymphocytes in Murine Small Intestine

Intraepithelial lymphocytes expressing &#947;&#948; T cell receptor (&#947;&#948; IEL) play a key role in immune surveillance of the intestinal epithelium. Due in part to the lack of a definitive ligand for the &#947;&#948; T cell receptor, our understanding of the regulation of &#947;&#948; IEL activation and their function in vivo remains limited. This necessitates the development of alternative strategies to interrogate signaling pathways involved in regulating &#947;&#948; IEL function and the responsiveness of these cells to the local microenvironment. Although &#947;&#948; IELs are widely understood to limit pathogen translocation, the use of intravital imaging has been critical to understanding the spatiotemporal dynamics of IEL/epithelial interactions at steady-state and in response to invasive pathogens. Herein, we present a protocol for visualizing IEL migratory behavior in the small intestinal mucosa of a GFP &#947;&#948; T cell reporter mouse using inverted spinning disk confocal laser microscopy. Although the maximum imaging depth of this approach is limited relative to the use of two-photon laser-scanning microscopy, spinning disk confocal laser microscopy provides the advantage of high speed image acquisition with reduced photobleaching and photodamage. Using 4D image analysis software, T cell surveillance behavior and their interactions with neighboring cells can be analyzed following experimental manipulation to provide additional insight into IEL activation and function within the intestinal mucosa.

We describe a method to visualize GFP-labeled &#947;&#948; IELs using intravital imaging of murine small intestine by inverted spinning disk confocal microscopy. This technique enables the tracking of live cells within the mucosa for up to 4 h and can be used to investigate a variety of intestinal immune-epithelial interactions. &#160;