Name: Assay for Pathogen-Associated Molecular Pattern (PAMP)-Triggered Immunity (PTI) in Plants (Video) | JoVE Journal
Uploaded: 2009-09-09T16:30:00.000+00:00
Duration: 8 min 45 s
Description: Learn a cell death-based assay for PAMP-triggered immunity in Nicotiana benthamiana plants. This method evaluates PTI responses using bacterial infiltration and helps researchers study plant defense mechanisms.

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5 minutes

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°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°C.</li>
</ol>
DAY 2:
<ol start="2">
<li>For Pseudomonas, add 150-200 μl 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 μM acetosyringone and let it grow overnight to 20 hours at 250 r.p.m., 30°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°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 –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 ΔhopQ1-1 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 ΔhopQ1-1 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="Plant pest detection, insect movement on leaf, comparison diagram, circled area with red arrows." border="0" data-altupdatedat="1787630408000" data-alt="Figure 1"> 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 ΔhopQ1-16</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 ΔhopQ1-1</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/pubmed?term=((Bacterial+elicitation+and+evasion+of+plant+innate+immunity%5BTitle%5D)+AND+%22Nat+Rev+Mol+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((The+plant+immune+system%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Bacterial+disease+resistance+in+Arabidopsis+through+flagellin+perception%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">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/pubmed?term=((Nicotiana+benthamiana%3A+its+history+and+future+as+a+model+for+plant-pathogen+interactions%5BTitle%5D)+AND+%22Mol+Plant+Microbe+Interact%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Plant+J%22%5BJournal%5D)">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.

<table><tbody><tr><td>MES</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>Beckman Coulter Inc.</td><td>DU 730</td><td></td></tr></tbody></table>

assay for pathogen-associated molecular pattern (pamp)-triggered immunity (pti) in plants

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.

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 ...

Hi, I am Suma TI from the lab of Greg Martin at the Voice Thompson Institute for Plant Research. Today we will show you a procedure for a cell death based assay for PAM triggered immunity or PTI. We use this procedure in our lab to look for the involvement of a gene in PTI.So let's get started. Begin the procedure by growing Nicotiana ANA until they're about seven weeks old. Four to five days prior to starting.The PAM triggered immunity or PTI assay. Trim the plants to remove all axillary branches and flowers. Try removing the axillary branches soon after they emerge to make the plants more manageable.After preparing the plants proceed to grow the bacteria. This procedure uses non-pathogenic bacteria for induction of plant immunity prior to a second inoculation with challenge bacteria, initiate growth of all the bacteria used in the assay. At the same time, use frozen glycerol stalks of the pseudomonas bacteria for each pseudomonas bacterial stalk.Spread a small amount of bacteria on the center of A KBM plate containing the appropriate antibiotic and incubate the plate at 30 degrees Celsius for about 18 to 24 hours. For the agrobacterium, use a previously prepared fresh plate to start a liquid culture in two milliliters of lb. Grow overnight at 30 degrees Celsius with shaking on the following day.Add 150 to 200 microliters of liquid KBM to the plates of the pseudomonas bacteria. Spread the bacteria over the entire plate. Using a sterile greter.Put the plates back in the incubator for another 20 to 24 hours. Four agrobacterium. Start a secondary culture in about five to 10 milliliters of LB containing 20 micromolar acetone.Grow the culture overnight at 30 degrees Celsius with shaking. After preparing the bacteria proceed to prepare the plants for the assay the day before conducting the PTI assay. After preparing the bacteria, move the plants to a 22 to 24 degrees Celsius room with approximately 35 to 40%relative humidity and constant light.Next, use a thick black marker to mark circles on the leaves. Choose well expanded leaves and avoid older leaves or leaves that are tough or thick to the touch. Avoid marking the lower portions of the leaf.The circles are at least 1.5 centimeters in diameter and two to four circles can be marked per leaf. The circles should be well spaced and preferably not cross a large vein. Start the PTI assay by harvesting the bacteria used in the induction of PTI.The pseudomonas species used should have formed a bacterial lawn indicative of good growth. Harvest them from the plate by scraping off with a sterile long wooden stick and resuspend in about 20 to 25 milliliters of 10 millimolar. Magnesium chloride solution for agrobacterium centrifuge.The culture to collect the cells and resus suspend in about 20 to 25 milliliters of 10 millimolar magnesium chloride plus 10 millimolar. MES pH 5.6, repeat the centrifugation and again, resus Suspend in about 20 to 25 milliliters of the magnesium chloride plus MES solution. Now that the inducing pseudomonas and agrobacterium bacteria are resuspended.Measure the optical density of all three cultures at a wavelength of 600 nanometers. Adjust the ODS by diluting the cells with magnesium chloride for the pseudomonas and MES plus magnesium chloride for the agrobacterium. The final required OD values are found in the accompanying written protocol.A total volume of 25 milliliters of bacterial culture is sufficient to inoculate about 40 to 50 spots. Now start the assay by infiltrating the PTI inducers into the pre-marked circles on the leaves using a one milliliter syringe without a needle. Write down the order in which the plants were infiltrated and the exact time when the infiltration was begun following the induction.Proceed to the challenge inoculation several hours after the induction inoculation. Harvest the bacterial strains required for the challenge inoculation as previously shown for the inducing pseudomonas strains. Again, measure the OD and adjust the concentration of the cultures according to the accompanying GR protocol.Start the challenge inoculation seven hours after the inducer infiltration. Use the following inducer challenger combinations. Choose a point on the periphery of the first inoculation circle as the center of the second inoculation circle.Perform the challenge infiltration in the same order of plants as that of the induction. If there are multiple spots on a leaf, then make sure the infiltrations do not overlap. Finally, plate serial dilution of all the cultures used in the assay on KBM or LB plates containing the appropriate antibiotics.Two days after the challenge inoculation one can start evaluating the PTI response two days after the induction and challenge inoculations look for the appearance of cell death due to effector triggered immunity or ETI in the spots that were challenged with PST DC 3000 cell death inside the overlapping area of infiltration indicates a breakdown of PTI and should be scored as a positive phenotype four to five days after the induction and challenge inoculations look for the appearance of cell death due to disease in the spots that were challenged with the DC 3000 hop Q1 mutant or PS back high again. Look for breakdown of PTI that is indicated by cell death due to disease in the overlapping area of infiltration. Continue to observe the plants until those which are not compromised for PTI begin to show cell death in the overlapping area of infiltration.Plants silenced for FLS two are compromised for PTI two days after inoculation with the PFDC combination. Cell death is seen in the overlapping area indicating breakdown of PTI control plants that have not been silenced for any gene. Were also inoculated with the PFDC combination and examined two days after conducting the assay cell death due to the challenge is seen outside but not inside the overlapping area due to a normal PTI response.We've just shown you how to perform a cell death based assay for pant triggered immunity or PTI while doing this procedure. It's important to remember to keep the plants in the environmental conditions that we recommend. Higher levels of humidity would negatively affect the plants and make the assay proceed more quickly.So that's it. Thanks for watching and good luck with your experiments.

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

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Learn a cell death-based assay for PAMP-triggered immunity in Nicotiana benthamiana plants. This method evaluates PTI responses using bacterial infiltration and helps researchers study plant defense mechanisms.

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

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