By Emily Stewart
Healthy arabidopsis plants
This week in lectures we disccussed plants immune response to various pathogens, focusing mainly on bacteria. Pseudomonas syringae is a hemibiotrophic bacteria, so when it infects a plant, the plants follows biorophic immune pathways and will activate the hypersensitive response causing localized cell death. After the bacteria has multiplied to a certain concentration within the plant, it switches to necrotrophic pathways, where the cell death from the biotrophic pathways helps the infection progress.
In the first lab this week, we infected four week old Arabidopsis thaliana plants with Pseudomonas syringae. The technique is important for this procedure so we practiced on used Arabidopsis plants with water before innoculating the plants that were used for the experiment. We used needleless 1ml syringes to force a magnesium chloride solution containing the Pseudomonas syringae (from cultures made the previous week) into the stomata on the underside of the fourth and fifth leaves of the plants. The plants were then covered and put in a growth room for two days.
Innoculating Arabidopsis thaliana Innoculated plants in the growth room
In the second lab, we obtained samples from the infected leaves and made bacterial cultures from them. To untrained eyes it was hard to identify the infected leaves, so it was fortunate that we had marked them before hand. After homogenizing tissue from each plant and magnesium chloride solution, we made six 10x serial dilutions. We then dropped 20µl of each dilution onto a KB-Strep plate. After incubating for two days, we were able to count individual colonies in some of the dilutions. Using the number of colonies in a spot on the plate, we can approximate how much bacteria is present in the plants.
Homogenized tissue samples Serial dilutions
Bacterial culture plate
All of the infections were pretty successful. Most of the infected leaves showed a pseudomonas syringae growth logarithm between 1.E+06 and 1.E+07. Two peoples plants showed slightly less bacterial growth than the others and had a growth logartithm slightly less than 1.E+06 and one person’s plants showed a growth factor a bit higher than 1.E+07.