By Sarah Coffee
We have begun to work with arabidobsis thaliana! But first, we learned about how plant infection works.
There are only eight genera of bacteria that infect plants. In our case, we infected the poor plants with pseudomonas syringae. We learned in lecture how bacteria usually enter through open wounds or natural openings in the plant, such as the stomata. To help the bacteria out, we used flat headed syringes to physically push the serum containing bacteria into the leaf stomata. We chose (and marked) two leaves on each plant that were not too old and not too young, and flipped them over and injected. If injected slowly enough and the right way the leaf will darken as it absorbs the serum. This is a delicate art in itself, so we tried (and failed) with normal water first.
Once they were infected we placed them in the plant room to allow the pseudomonas to grow for a few days. Bacteria thrive in warm and moist conditions. While the bacteria grew, the plant began its defenses, represented with the Zig-Zag Model. It starts with PTI, or Pattern-Triggered Immunity. This is where the plant uses its PRRs (Pattern Recognition Receptors) which are normally leucine rich, repeat receptor kinases. These structures recognize highly conserved elements of the bacterial genome, such as a section of the flagellum that all bacteria have. These PAMPs, or pathogen associated molecular patterns, activate the plant defenses through PTI. However, the bacteria will then release effectors that turn off PTI, resulting in Effector-Triggered Susceptibility. The plant fights back using Effector-Triggered Immunity (ETI), where the plant may recognize some of the pathogens receptors. This game of cat and mouse can go either way, with both parties constantly evolving. Something as simple as environmental conditions may sway the outcome of plant vs pathogen!
The following Thursday we retrieved our plants and began cutting out small circles from the leaves that were infected. (pic1-pic3) Besides having been marked, infected leaves could be determined from the yellowing of the leaves, marking spots where the plant had fought back against the bacterium.
In lecture we learned about biotrophs and necrotrophs which all use different methods of infection. The bacterium we used today, pseudomonas syringae, is a biotroph. This means that the plant will respond with the stress salicylic acid; if it were a necrotroph it would use jasmonic acid. Crosstalk between the hormones prevents both from being activated at the same time; if faced with a choice between the two, the plant will produce Salicylic acid defenses. Salicylic acid triggers the Hypersensitive Response (HR) that results in the production of ROS (among other compounds). The ultimate result is that the plant undergoes necrosis of its infected cells in order to quarantine the bacteria to those few spots; this is what causes the yellow spots we see on the leaf. While this plant looks sick, it is still alive and fighting back.
Once we cut out our pieces we put them in grinding tubes and sent them to the homogenizer. (Pic 4) We then made several dilutions of this bacterial solution and placed these onto agar plates (pic5 and pic6).We diluted so that at some level the pseudomonas would be in individual colonies, making it easier to count with the naked eye.
Where did we get these plates? Well we made them ourselves in Lab #2! We split up into four groups with different medium recipes: YPD (modified), V8,YDC (modified), and KB solid medium. After mixing all the ingredients together, we autoclaved the solutions. We then cooled them, stirred them, and voila! They were ready to pour out onto the plates. After about an hour the plates were ready to bag and store for the next lab!
Back to bacteria: the following Tuesday we counted up our colonies. (pic7) Using the dilution number, we figured out how many bacteria had successfully grown inside our plants. All of us had very sick plants!