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BS32020: “Revision” Workshop
Name………………………………
This workshop report is designed to supplement your lectures on bacterial
pathogenesis and systemic acquired resistance. The demonstrated experiments have
been designed to follow a logical progression and as you complete the questions
below you are in fact writing the outline of a paper.
Experiment 1: Resistance versus Susceptibility
A.
Variable pathogen genotypes. Go to bench 1B. You will find Samsun plants
which have been infected with Pseudomonas syringae pv. (P. s. pv. ) phaseolicola, P. s.
pv. phaseolicola hrp L mutant; P. s. pv. tabaci and P. s. pv. tabaci tabtoxin- mutant.
Infected plants have been designated A, B, C and D. Within each group of plants were
infected 6, 24, 72 and 144 hours ago (designed A6, A24…etc).
In this part of the experiment you will use your newly acquired knowledge of plant
pathology to deduce what A, B, C and D have been infected with. This should be based
on symptomology, bacterial population and gene expression data (the latter two results
are also to be found on bench 1B).
1. Describe the symptoms and their speed of development in
Infection A
Infection B
Infection C
Infection D.
2. Look at the bacterial population data. Describe the trends you are observing.
Infection A
Infection B
Infection C
Infection D.
Look at the accumulation of PR1 protein as detected by Northern blotting. Describe
the trends you are observing.
Infection A
Infection B
Infection C
Infection D.
3. Based on your observations what do you deduce strains A, B, C and D to be?
Explain your conclusions.
Experiment 2: What is Systemic Acquired resistance?
Go to bench 2. You will find tobacco plants that have been infected in their lower leaves
with TMV (1o infection) and subsequently (i.e. after 7 days) in the upper leaves (2o
infection). You will also find plants that have been only infected in their upper leaves these are controls.
1. Measure ten lesions in the upper leaves of a control and SAR plant.
Mean results =
2. Based on your observations make in Experiment 1 - what do the think effect on
bacterial population would be if you were infecting with an avirulent and virulent
strains?
Look at the PR1 transcript accumulation as detected by northern blotting and SA
accumulation in systemic leaves following either mock- or real infection with TMV.
3. Describe your observations and conclusions.
Experiment 3: What is the systemic signal in SAR?
Go to bench 3: You will find Samsun tobacco with have been watered with 1mM SA, and
just water for four days before being infected with TMV.
1. Measure the lesion size of ten lesions on plants fed with the each chemical
Mean results =
Look at the PR1 transcript accumulation in SA-treated plants before infection with
TMV as detected by western blotting.
2.
What was the purpose of these experiments and what do you conclude?
Experiment 4: Can we prove that salicylic acid plays a role?
Go to bench 4. You will find wild type Samsun plants and a transgenic line where a
bacterial gene, salicylicate hydroxylase (SH-L), has been introduced. SH-L is an enzyme
which converts salicylic acid to catechol. Each plant has been sequentially infected with
TMV as outlined in Experiment 2.
1. This time, measure lesion sizes in both 1o and 2o infected leaves. Compare with
measurements of equivalent lesions from SAR exhibiting wild type Samsun plants
(Experiment 2). What was the purpose of this experiment? What do you conclude on the
role of SA in both 1o and 2o secondary lesions?
Experiment 5: How does salicylic acid confer increased resistance?
Another way to measure gene expression is to use “reporter genes”. An easily assayed
biomarker is fused to the regulatory region of a gene. This can give you quantitative data
which is an indirect measure of gene expression. This can be a very important tool in
working out how a chemical signal may be acting.
Below is reporter gene activity following SA treatment of two different transgenic lines.
To help you interpret this data, AoPR1 –Reporter is responsive to hydrogen peroxide.
+ water
+ SA
+ TMV
+ TMV/SA
PR1-Reporter
253
4100
2190
5342
AoPR1-Reporter
102
132
781
3121
Interpret the above data. What does this tell you about the role of SA in HR and SAR?