The OUTPACE program allows students to enter the world of plant pathology and seek to further understand the complex process of plant disease resistance. Lectures on bacteria introduced the process of defense that plants go through while in attempt to combat bacterial infections including gene regulation and a hypersensitive response. Bacteria Pseudomonas syringae is a bacteria that infects a very wide range of common plants, one which most have seen but may not fully understand. Details about the infection have been studied and are reported by many different scholarly articles that can easily be found online. Bacteria and other pathogens usually infect through a vulnerable area, such as a physical wound, to begin the infection process. Once inside the plant, invaders use many different defensive strategies to sneak its way out of being killed off.
This assignment followed two lab sessions in which Arabidopsis plants were infected with Pseudomonas syringae, bacterial cultures were grown, and then colonies were counted. The experiment began with small Arabidopsis plant leaves being infected with a needless syringe via the stomata on the bottom side of the leaf. The forcing of the liquid holding bacteria made it more likely that the leaf would become infected compared to any other pathway. Two type of Arabidopsis plants were used, including a npr-1 plant that is genetically mutated to be disease susceptible, and the Col-0 plant that is disease resistant. Theoretically, there should be more evidence of bacterial infection on the npr-1 plants than the Col-0 plants show.
Two days after the Pseudomonas injection the plants were revisited and proof of infection was limited at best. Most plants did not seem sick, and the infected leaves were only noticeable because they were labeled beforehand. The process to grow bacterial cultures began with obtaining samples from both the npr-1 and Col-0. Eight samples of each type were homogenized and then the sample was diluted. After obtaining the two dilutions, the two cultures were now ready to be grown on KB plates.
Four vials of five dilutions from each plant type were grown on two plates that allowed for a total of 20 colonies to be observed for each person.
This experiment was concluded after the most dilute colony was counted and the data was entered into an excel spreadsheet pre-fromulated to graph results from our npr-1 compared to our Col-0 colonies. Only one person had ideal results, and the rest of the colonies had not had time to fully develop due to severe time constraints. The colonies will be revisited in a few days and hopefully better outcomes will be present. Overall, the experiment was very fun, a little frustrating (due to small plants), and rewarding. Our class got to practice valuable lab skills and we were all very thankful to learn to grow bacterial cultures.