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Lab exercise outline	lab exercise for print-out Slideshow of Experiment
In Module 1 - Lab, you showed that chrysanthemum is resistant to Pseudomonas syringae. In this Lab Exercise you will test two different tomato cultivars to show how some can defend themselves against P. syringae while others cannot.

Introduction

Experimental Objectives

Materials

Experimental Procedures

Results

Questions

Introduction: How do plants fight disease-causing microorganisms
Plants have powerful ways to fight fungi, virus, bacteria and parasitic worms that can cause plant disease. They have preformed barriers, such as waxes on the surface of their leaves, or the cell wall that acts as a shield for the plant cell.  And they also have a very effective immune system. This system is induced in plant cells when these disease-causing microorganisms, referred to as PLANT PATHOGENS, attempt to attack them and it involves very sophisticated molecular mechanisms.

The plant immune system consists of two different pathways. One is activated within minutes of attack and is called PAMP-triggered immunity (PTI). The other is activated a few hours after pathogen attack and is referred to as effector-triggered immunity (ETI). Here we will describe the way these two pathways work in response to attempted infection of plants by bacterial pathogens.  Similarities also exist with what happens when fungi and some other pathogens attack plants.

The PTI response pathway (also called ‘basal defense’) is activated by molecules commonly found in pathogens.  These molecules are called PAMPs, for Pathogen-Associated Molecular Patterns. PAMPs often have an essential role for the life of the bacteria.  For example, they can be components of the bacterial membrane, or a component of flagella, structures that enable the bacterium to move in its environment.  Bacteria therefore cannot get rid of these molecules and yet they act like flashing lights that give the bacteria away because plant cells have proteins specialized for recognizing PAMPs.  These proteins are called PRRs, for Pattern Recognition Receptors.  A plant PRR is able to detect a certain PAMP (e.g. flagella) from many different bacteria – whether or not it is a pathogen. Once a PAMP has been recognized by the corresponding PRR, a signaling pathway is launched in the plant cell activating defense mechanisms.  For example, the plant cell wall is strengthened and plant cells produce antibacterial compounds. 
 
This first immune system is not always sufficient to stop a bacterial infection.  As you have learned in Module 1, in order to invade plants, plant-infecting bacteria often use a strategy shared by numerous disease-causing bacteria, including some responsible for human infections such as the bubonic plague.  This strategy relies on a molecular device that looks like a syringe. The molecular syringe, referred to as the type III secretion system, is synthesized by the bacterium when it makes contact with the host cell it is trying to infect.  The type III secretion system allows the bacterium to inject a large number of different proteins directly into the host cell. The injected bacterial proteins act like sophisticated sabotage weapons intended to alter cell metabolism.  These injected proteins are referred to as ‘virulence proteins’ or more generally as EFFECTOR PROTEINS or ‘effectors’.

Scientists have known for a long time that without a functional molecular syringe, the bacterium fails to cause disease. Studies now aim to understand the specific role of each effector. Recent results revealed that several effectors can suppress the plant basal defense, allowing disease to occur

Because bacterial effector proteins are so important to the pathogen’s ability to cause disease, the plant has evolved a way to also detect these proteins. This second immune system is referred to as effector-triggered-immunity (ETI) because it is activated by effectors once they are injected into plant cell. 

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ETI is more specific than PTI because it detects specific proteins injected by the pathogen rather than PAMPs which are common molecules expressed by all microbes.  For ETI to occur it requires that the plant express a resistance protein that specifically detects a certain effector protein.

If both the effector and the resistance protein are together in the plant cell, defense mechanisms are activated and trigger a HYPERSENSITIVE RESPONSE (HR) similar to the one you’ve learned about during Module 1. The plant cells die, which blocks further bacterial growth, and the disease can’t develop. The plant is said to be resistant to the bacterium.

Plants use programmed cell death (PCD) to create a protective zone of dead cells (brown) around the site of pathogen invasion (purple). The dead plant cells do not support viral growth and lose their interconnectedness, thereby halting the pathogen's spread. Credit: Nicolle Rager Fuller, National Science Foundation Source: Science Daily

Our understanding of ETI is based on work by scientists in the 1940s which led them to propose the gene-for-gene model of plant immunity.  This model is based on the observation that some varieties of a particular plant species express a resistance protein while others do not. 

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For simplicity, let’s say that ‘R’ is a plant resistance gene and ‘r’ indicates that the plant lacks this gene.  Similarly, a particular pathogen may express a certain effector gene or it may lack that gene.  Again, for simplicity let’s say the effector gene is designated as ‘A’ and the lack of the effector gene is designated as ‘a’.  The gene-for-gene model states that when a plant expresses R and the pathogen expresses A then recognition occurs, ETI is activated, and no disease is formed.  However, if the plant doesn’t have R, or the pathogen doesn’t have A, then recognition does not occur and the pathogen is able to escape detection and cause disease.

2. Experimental objectives

You will work with two Pseudomonas syringae pv. tomato strains (Pst1 and Pst2) and two tomato varieties (A and B).

Your goal is to identify the tomato variety (A or B) that can specifically resist infection by bacterial strain Pst1 and to interpret your results in the light of the ‘gene-for-gene’ model of plant immunity.

The experiment consists of dipping tomato plants into bacterial suspensions and observing development of disease or resistance.

Watch the Slideshow of the Experiment

3. Materials:

Two 4-week-old tomato seedlings A
Two 4-week-old tomato seedlings B
(The plants are covered with plastic bags the day before the experiment to keep a high humidity level)
Two 2-liters beakers (or 1 gallon plastic milk jugs) 
Two empty plastic tubes of 1.5 ml
Two plastic tubes of 1.5 ml containing a surfactant (labeled ‘S’)
Two plastic pipettes of 1ml
One 1-liter cylinder (or 1 liter empty plastic bottle)
Two flat wooden toothpicks
One marker
Two 10” glass stir rods (or long spoons)
2 square of aluminum foil
Paper towels
Tap water
One timer or clock

*To share with another group:
Petri plate with freshly grown bacteria (Pst1 strain)
Petri plate with freshly grown bacteria (Pst2 strain)

NOTE: An additional experiment, consisting of dipping plants in a suspension without bacteria, will be done for the class by your teacher or one group of students, following the exact same protocol.  Make sure you are aware of the results of this experiment.

* Materials for the additional experiment:
Two 4-week-old tomato seedlings A
Two 4-week-old tomato seedlings B
One 2-liters beaker (or 1 gallon plastic milk jugs)
One 1-liter cylinder (or 1 liter empty plastic bottle)
One 10” glass stir rod (or long spoon)
One plastic tube of 1.5 ml containing surfactant (labeled ‘S’)
One plastic pipette of 1ml

4. Experimental procedures

ATTENTION: You must be careful how you handle your tools, dishes and wastes. You don’t want to cross-contaminate your experiment by mixing Pst1 and Pst2.

*Preparation of the bacterial suspensions

You will need to prepare:
‘Pst1 suspension’: will contain water, surfactant and Pst1 bacteria
‘Pst2 suspension’: will contain water, surfactant and Pst2 bacteria.

1. Label one beaker with ‘Pst1’.
2. Using the 1-liter cylinder, pour 2 liters of tap water into the beaker.
3. Add the surfactant to the beaker full of water.

A surfactant is added to the bacterial suspension to break the natural surface tension of the leaf so that the bacteria do not just wash off the leaf after dipping

4. With a disposable plastic pipette, add water into a 1.5ml plastic tube up to the 1ml line.
5. With a flat wooden toothpick, scratch out bacteria from the Petri plate containing the freshly grown Pst1 strain (be gentle, take only the bacteria, not the medium on which the bacteria are growing). Take enough ‘bacterial paste’ to obtain a ball the size of half a green pea (see below).
6. Resuspend bacteria on the tube containing 1ml of water.  Mix carefully with the toothpick to obtain a homogenous suspension.
7. Repeat 3 more times.

Try to obtain a ball of about this size:   and resuspend this ‘bacterial paste’ in the tube with water. Do this 4 times in total (it should correspond to about half the Petri plate).

NOTE: You will need to add the same amount of bacteria to each suspension (Pst1 and Pst2) in order to have comparable results. In a lab, you would use a spectrophotometer to determine the optical density (OD) of your suspension. The OD would allow you to determine the bacterial density of your suspension so that you can dilute it if needed. You also need to prepare a suspension at a bacterial density known to cause disease on susceptible plant. In a lab, you would have checked published papers to learn what concentration scientists used in their experiments and/or you would have tested several concentrations to obtain one that causes disease. Here, we have determined the concentration to use for you and we have estimated the volume of ‘bacterial paste’ to which it corresponds, so that you don’t need a spectrophotometer.

8. Using the disposable plastic pipette, take the concentrated bacterial suspension you just prepared and add it to the bucket containing water + surfactant.
9. Mix well using one long spoon.

*Plant inoculation

You will need:
One plant A and one plant B for dipping in ‘Pst1 suspension’
One plant A and one plant B for dipping in ‘Pst2 suspension’

10. Label one plant A and one plant B with Pst1
11. Use a piece of foil to fold over the top of the pots to prevent the soil from falling out when you turn the plant upside-down. Turn your plant A labeled Pst1’ upside down and dip it into the suspension for 2 minutes (use a timer or your clock). While dipping, gently move the plant up and down (be careful not to break branches).
12. After 2 minutes, take the plant out of the suspension, remove the foil, and lay it on a paper towel to let it dry about 5 minutes.

Make sure the suspension is mixed with the spoon right before dipping to resuspend bacteria that are likely to have fallen to the bottom of the bucket.

13. Repeat the same process with your plant B labeled Pst1.
14. Water the plants, wrap them in a plastic bag and put them back near the window
15. follow the exact same procedure for ‘Pst2 suspension’

Be careful to wash your hands after you worked with Pst1, and to use new tools and a new piece of foil.

16. After one day, take off the plastic bags and keep watering the plants once a day.

5. Results

5-6 days after dipping the plants:
-Observe your Pst-dipped plants and the plants dipped only in the suspension without bacteria.
-In the chart below: Write ‘Disease’ if you see disease symptoms on plant leaves, or ‘Resistance’ if you don’t see any symptoms.

Compare with other lab groups’ results.

You didn’t have a chance to do the lab activity?
No worries! You can watch here what you would have seen.

6. Questions

1. Why does one of the treatments consist of dipping tomato plants in a suspension without bacteria?

2. Why do you compare your results with other lab groups?

3. Which plant is resistant to Pst1 strain, plant A or plant B?

4. How can we tell that the resistant plant is specifically resistant to Pst1?

5. In light of the ‘gene-for-gene’ model of plant immunity presented in the introduction:

a) Explain what the results suggest about the genes/proteins of the bacterial strain Pst1 and about the genes/proteins of the resistant plant.

b) Explain what the results suggest about the bacterial strain Pst2.