Module 1 Home	Biological Lab	Social Science Issue	Process of Science	Web Exercise
Lab Exercise Outline
In Module 1 Home, you learned that Pseudomonas syringae can cause diseases on agriculturally important crops such as tomato. In this lab exercise you will test whether a different plant, chrysanthemum, is susceptible or resistant to P. syringae

Introduction What is Pseudomonas syringae? Why are some plants resistant and some susceptible? Summary Lab Exercise - Section A Experimental objectives Materials and methods Results Post lab questions Lab Exercise - Section B Experimental objectives Materials and methods - Materials - Experimental design - Experimental procedures - Experimental setup Results Post lab questions
experimental technique: making dilutions and estimating area	lab exercises for print-out

Introduction
What is Pseudomonas syringae?

The bacterium Pseudomonas syringae causes bacterial speck disease on tomato plants and causes significant crop loss every year. The symptoms of bacterial speck disease are small brown lesions on tomato leaves. The bacteria are spread from plant to plant by water splash and enter the plant through the stomata. The species P. syringae is divided into pathovars because the host range of individual bacteria can vary. The pathovar designates the host plant and is indicated by the pv following the species name. For instance, P. syringae pv. tomato, which you are working with today, infects tomato plants. Most other types of plants are resistant to this pathovar.

Like people, individual plants respond differently to disease-causing bacteria. When P. syringae lands on a plant that is SUSCEPTIBLE, disease develops. P. syringae is not able to infect every plant because some are RESISTANT. They are resistant because the plant realizes early on it is being infected and triggers a HYPERSENSITIVE RESPONSE (HR). The hypersensitive response is suicide of the infected cells in order to stop the pathogen from spreading. It essentially cuts off the bacterium's food supply. HR results in brown lesions (due to dead cells) on the leaf that look similar to disease, but are actually a form of plant defense. Though HR may seem strange because the plant is killing its own cells, it is analogous to unpleasant human responses to infection, such as fevers, rashes, and allergic reactions. Plants do not have an immune system like the one in humans and have different ways of fighting off pathogens. They also release antimicrobial factors and strengthen their cell wall when they are being infected.

Why are some plants resistant and some susceptible?

When bacteria try to infect a plant what is actually occurring is a "war of molecules". Disease or resistance is brought about by a series of complex interactions between pathogen and host molecules. When Pseudomonas syringae infects a plant it injects many different proteins, called EFFECTORS, into the plant cell. Effector proteins are very diverse and help the bacteria cause disease in the plant. To inject effectors, P. syringae uses a highly specialized apparatus called the TYPE III SECRETION SYSTEM. The type III secretion system works like a syringe to inject effectors directly into the plant cell. This system is used by many bacterial pathogens, such as the bubonic plague pathogen Yersinia pestis, to infect their hosts.
If special plant molecules, called RESISTANCE PROTEINS, are able to recognize the "foreign" bacterial effectors, the plant will know it is being infected and HR will be triggered. This will kill the bacteria and the plant will be resistant. If this recognition does not occur, the bacteria can grow normally and disease will develop.

What happens when P. syringae infects a…

Susceptible Plant	P. syringae can infect these plants and cause bacterial speck disease. It injects molecules called effectors into the plant that cause disease.
Resistant Plant	P. syringae cannot infect these plants. The plant is resistant due to the HR defense. The plant turns on HR because it has resistance proteins that recognize the bacterial effectors. This recognition tells the plant it is being 'invaded' and that it should 'arm' itself.

Summary:

• P. syringae pv. tomato causes bacterial speck disease on susceptible hosts, such as tomato.
• P. syringae pv. tomato causes the hypersensitive response (HR) on resistant hosts.
• A hypersensitive response is suicide of the infected cells in order to prevent the pathogen from spreading.
• P. syringae injects effector molecules into the plant cell to cause disease.
• Plants have resistance proteins that recognize effectors and turn on the HR response.
• If the plant does not have the correct resistance proteins to recognize the effector, disease will develop.
  

Plants have different sets of resistance proteins and P. syringae strains have different effectors, so in certain combinations disease still occurs. Current research is focused on the specific interactions between effectors and resistance proteins and how this can lead to increased disease resistance. In the Pseudomonas-Plant Interaction Project, one goal is to use genomics to identify all of the effector molecules made by P. syringae.

Lab Exercise - Section A

Experimental Objective

Inoculate leaves with Pseudomonas syringae pv. tomato and Pseudomonas fluorescens and evaluate the different responses.

Materials and Methods

Work in groups of 3

Materials:

1 Leaf *
1 Beaker
Bacterial culture plates (P. syringae pv. tomato and P. fluorescens)
Graduated Test tubes (~10-15 ml) (~6-8/group)
Water
Disposable culture loops
Marker
Dissecting needle
Plastic pipette droppers
Turbidity Standard

* This can be one tobacco leaf or a cutting with three chrysanthemum leaves

Methods:

1. Cut off one large leaf or several small leaves from the plant provided. Place in water in a beaker or cup.

2. Label three graduated test tubes: P. syringae, P. fluorescens, or water

3. Add 10 ml of water to each test tube

4. Using a disposable loop, scrape some bacteria from the plate labeled P. fluorescens. Shake off the cells in the tube labeled P. fluorescens. *Remember to dispose of loop in autoclave bucket after each use!

5. Vortex tube to un-clump cells. If you don't have access to a vortex, pipette gently up and down with the plastic transfer pipette to break up the cell clump.

6. Adjust the density by adding more cells or water until you reach an OD600 = 1.0. OD stands for Optical Density, and is a measure of how much light of a particular wavelength (in this case 600 nM) a solution blocks. You can measure OD using a spectrophotometer or by comparing the bacterial suspension to a turbidity standard.

7. Repeat steps 4-6 for P. syringae

8. Choose six sections on the leaf (choose areas between veins). Label each section with a marker: W for water, Pf for P. fluorescens, or Ps for P. syringae (2 replicates for each)

9. With a dissecting needle, poke very small holes into the center of each section on the bottom of the leaf. (do not make the hole in a vein)

10. Suck up some P. fluorescens bacterial suspension with the plastic pipette dropper. Put your finger behind the hole you just made for support. Place the dropper on top of the hole and apply some pressure but not enough to break through the leaf. Squeeze the pipette. You should see liquid spreading throughout the inside of the leaf.

11. Repeat Step 10 for P. syringae and the water

12. Leave cuttings on the benchtop and examine at 48 hours

Results

Record your observations by words or drawings for:

P. fluorescens

P. syringae

Water

Post Lab Questions

1. What control is used in this experiment and why?

2. What is the hypersensitive response (HR)?

3. Why does P. syringae pv. tomato cause disease on tomato but HR on tobacco?

4. Do you think you would see HR in nature?

5. Why is P. syringae a good model system? Give an example of another model system.

6. What bacterium caused the black plague? How is the hypersensitive response and the black plague connected in a way that makes scientists studying each work closely together?

 

Lab Exercise - Section B

Experimental Objective

Determine the concentration of Pseudomonas syringae pv. tomato necessary to trigger a visible hypersensitive response in the test plant.

Materials and Methods

Work in groups of 3

Materials:

Leaves *
1 Beaker
Bacterial culture plates (P. syringae pv. tomato and P. fluorescens)
Graduated Test tubes (~10-15 ml) (~6-8/group)
Water
Disposable culture loops
Marker
Dissecting needle
Plastic pipette droppers
Turbidity Standard

* You will have to determine the number of leaves your group will need based on your experimental design. If you are using a plant with large leaves, such as tobacco, you can perform multiple infiltrations on one leaf. You will need more leaves (or entire stems) if you are using plants with smaller leaves, such as mums.

Experimental design:

The Hypersensitive Response (HR) that plants exhibit is a specific reaction to effector molecules injected by pathogenic bacteria. If the bacteria are present in small numbers, then a few cells in the leaf commit suicide. In this case, there is no change in the leaf that is visible to the naked eye. On the other hand, if an excessive number of bacteria are infiltrated into a leaf a large number of cells will commit suicide. It is possible that so many plant cells commit suicide that there are not enough cells remaining to maintain healthy tissues. In this case, all the cells in the infiltrated area die and a large brown-grey splotch appears on the leaf.
In this lab we are going to set up and conduct an experiment to determine how many bacteria it takes to cause the plant to exhibit a visible HR. In order to do this we will need to test different concentrations of bacteria to see if they cause a visible HR response.

Experimental procedures:

It is very important to plan out an experiment before actually starting it. Not only do you need to know how much materials you will need, but you also need to make sure that you will be able to understand your results. You will find a discussions of techniques that you may find useful for this experiment under "Experimental Techniques: Making Dilutions and Estimating Area" in the PPI Supplemental Reading Packet.

Experimental setup:

Discuss with your group how you want to setup your experiment before you begin. Record your experimental design below.

Dilutions of P. syringae to test:

Controls to test:

Total number of test/samples:

Number of leaves needed:

Results:

In the space below, design a chart that accurately displays the samples that you tested and the results of your experiment.
The chart should include the following information: Bacteria type, concentration, HR (Y/N), area of leaf affected.

Post lab questions:

1. What concentrations of P. syringae did you test to see if they exhibited a visible HR?

2. What factors did you have to take into account when you determined which concentrations of bacteria to test?

3. What concentrations P. syringae did you find exhibited a visible HR?

4. What controls did you use in your experiment, and what information did you get from them?

5. How did you estimate the areas of your HR lesions? What factors could introduce error in your area calculation?

6. Assuming that a leaf cell is a cube that is 30 µM in all dimensions, and that a leaf is 5 leaf cells thick. How many leaf cells were in the HR patch of the lowest concentration of bacteria that exhibited a HR?

7. How many bacterial cells did you infiltrate into that leaf? Since it is difficult to measure the exact volume you infiltrated, due to leaking, you can estimate the amount of bacteria infiltrated based on the size of the HR lesion. Assume that the area of the infiltrated bacterial solution matches that of the HR lesion, and was 50 µM thick. Also assume that a bacterial solution with an OD600 of 1 has a concentration of 1,000,000,000 bacteria/ml.

8. From the numbers you just calculated, how many bacteria per plant cell does it take to cause a visible HR to occur?

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