by Josh Knapp
Last time we were in the lab we inoculated our Arabidopsis plants with the Pseudomonas syringae bacterium. We had two genetically different Arabidopsis; a wild type columbia plant, and a columbia plant with a mutated NPR1 gene. The mutant plant was more susceptible to pathogenic infection because the NPR1 gene codes for a protein that is crucial to plant immune responses. Knowing this, it was safe to assume that our NPR1 plants would look a little bit worse than our wild type plants after they had sat for a few days, and for the most part they did! Below are the wild type columbia plants and the NPR1 plants.
Aside from just looking at the appearance of the infected plants, our goal today was to collect tissue samples from the infected leaves and isolate the syringae colonies within them. We expected to see a greater number of colonies from the NPR1 plants due to their lack of immune response.
To isolate the colonies, we first took two infected leaves from each Arabidopsis plant and punched a hole through them using a standard, classroom hole-puncher. The reason we did this was to make sure that the sample of leaf tissue collected from each plant would be constant, the size of the hole punched, regardless of leaf size. Two “circles” of leaf tissue were added into homogenization tubes that contained Magnesium chloride (MgCl) solution and metallic beads. It is important to mention that we made sure no homogenization tube contained two leaf samples from the same plant, every tube contained two “circles” from two different plants (of the same genotype) in order to ensure that the results would be an average for all of the plants of that genotype. This prevented any one plant from drastically skewing the data if it had unusual results.
We then took the samples to be homogenized for six minutes on the highest setting! The homogenizer is basically a machine that just shakes the tubes very rapidly, but because we put the metallic beads inside of the tubes, the beads acted as grinders that broke the leaf tissue down completely.
After the samples were well mixed, we added additional MgCl solution to each tube to help dilute the mixtures somewhat.
However, we needed to make further dilutions of our bacteria to in order to isolate individual colonies. Our plan was to make a series of six dilutions for each sample tube, and because we had homogenized twelve sample tubes, six containing wild type tissue and six containing NPR1 tissue, we had to prepare 72 wells of MgCl2 solution for the dilutions! Unfortunately supplies were tight, so we couldn’t use a multichannel pipette for this part of the experiment and had to prepare each well the old-fashioned way, by hand.
After the dilutions had been carried out, we transferred 20 μl of the contents from each well onto the large agar plates we had prepared last week. The wild type and NPR1 were kept separate throughout the experiment so we ended up with two agar plates that each contained 36 “drops” of solution, each drop representing the contents of a well from our series of dilutions. Next time we come to lab, we expect to see each sequential drop contain fewer and fewer colonies due to having a lower bacterial concentration. Hopefully, some of the drops will have so few colonies
that we can easily identify and count them, allowing us to see whether more bacteria grew in the NPR1 or wild-type Arabidopsis plants.
UPDATE: Monday, May 21st
When we came back to the lab today we counted the number of colonies that grew on the agar plates. To count the colonies we looked at each of the different “columns” of bacteria, where each sequential “drop” in a column contained a lower bacteria concentration due to our dilutions. We determined which drop had the lowest number of colonies and counted how many colonies had grown in that drop. If there were only one or two colonies in the lowest concentration drop, we would not count that drop and would instead move to the previous drop in the column. We did this because while one or two colonies could have resulted from a very diluted concentration of bacteria, one or two colonies also could have grown randomly, due to some error, so we considered it best not to count any single or double colonies.
On the top is the columbia plate; unfortunately the plate was wet and many of the drops smeared, but it was possible to collect data for five out of the six columns. On the bottom is the NPR1 plate; it has more clearly defined colonies that were easy to count. Additionally, the comparison between these plates highlights how the columbia plants had fewer colonies overall as the dilutions continued.
After counting the lowest number of colonies from each column, we put the data for both the wild type and NPR1 agar plates into an excel spreadsheet to compare them. The spreadsheet calculated an average concentration of bacterial growth for each plate, indicating which plant had more bacteria growth overall. As predicted, NPR1 showed higher amounts of bacterial growth, indicating that the mutated plants did have a lower immune response compared to wild type Arabidopsis columbia.