Pseudomonas syringae infection

Earlier in the OUTPACE program, we learned the basics behind how an infection works its way into the plant and how the plant combats this infection through various methods including gene related responses as well as a severe response that could kill the infection along with the affected areas. The Pseudomonas syringae infection seems to be a fairly common one in the plant world as a quick google search yields plenty of scholarly articles detailing the infection. Bacteria and viruses normally work to find the easiest place of entry into a plant, most often settling for invading through a weak point such as a physical wound. The invaders will then work against the plant’s immune system in a variety of ways including disguising itself so that the plant can’t destroy the invading species.

This week we worked on infecting plants with Pseudomonas syringae, creating cultures of these bacteria, and finally counting the colonies at the end of the experiment. On Monday we prepared the plants and infected them by injecting the bacteria using a syringe without a needle on the underside of two leaves that were marked on each plant. We were told to be careful and not damage the leaf because the stress this causes and the response from the plant cells could actually weaken the immune response and make the plant more susceptible to infection. We used NPR-1 plants which were genetically mutated to have a weak or no immune response to the disease. The other plants used were COL-o, which was resistant to infection. In theory the NPR-1 plants should have been much more infected than the COL-o plants but this was not the case for everyone including myself.

NPR-1 and COL-o plants

NPR-1 and COL-o plants

On Wednesday we revisited our plants and no noticeable infection was noted on any of mine. Both types of plants looked very similar to what a normal plant would look like. On this day we worked to prepare samples of the infection for counting on Thursday. The procedure was fairly basic and included punching holes in the marked leaves, two for each sample that would be later homogenized. In total each member gathered 8 NPR-1 leaves and 8 COL-o leaves and put two into each vial and this was later homogenized and returned to us. Once returned, we proceeded to do serial dilutions and then dropped the dilutions onto a plate for each NPR-1 and COL-o plants. We used two plates and did 5 dilutions for each, and using the 4 vials, each person had 20 colonies to look at by the end of the experiment.

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Rajan working diligently on his plants (note the intense concentration)

 

 

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Rajan showing how it’s done when infecting the plants

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Infected plant (very difficult to tell)

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Working on the serial dilutions

 

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Finished culture waiting for it to dry (incorrect use of pipette resulted in colonies much larger than was necessary)

 

On Thursday we concluded the experiment by counting the most dilute colony we could see on each row. This data was entered into a specific spreadsheet that Dr. Mukhtar had created and the results were organized into a graph. Learning the skills required for this experiment was very rewarding and I feel that with more practice, my results would have been much better. Unfortunately, coming from the chemistry department, I had no experience using the pipettes that were used for biology labs and I used it incorrectly. This was corrected too late however and my results suffered and I actually got the opposite of what was expected, which was that the resistant COL-o had actually more colonies growing than the NPR-1 non-resistant species. At least now I know what  I did wrong and am able to go back and correct it next time. I enjoyed the opportunity to learn these experimental methods, which normally would not have been offered in any other class. We also were told that the experiment was rushed due to our time constraints and normally we would have taken more time to allow the colonies to develop.

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Clearly the COL-o has more visible colonies which is incorrect but the results can’t just be thrown away just as in any experiment

 

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