Weekly post by Maggie McCormack

We ended last week by inoculating our Arabidopsis thaliana plants with the hemibiotrophic bacterial pathogen Pseudomonas syringae. We used two genotypes of Arabidopsis–the wild type, Columbia-0, and a mutant lacking an important gene required for pathogen resistance, npr1. After the weekend, we spent a few days analyzing the results of the infection.

Visually, the two genotypes showed a marked difference in the level of infection. As expected, the resistant wild type plants looked healthier than the devastated susceptible plants, which had several wilted, dead leaves. We learned several lab techniques that allowed us to quantify these results into mathematical, scientifically usable data.

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First we took random, equivalently-sized samples from the leaves of both plant genotypes using a regular hole puncher, suspended them in solution (MgCl2), and ground the leaf discs until the mixture was homogenized. Then we diluted the bacterial concentration and pipetted the solution onto KB plates.

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A few days later we were able to count individual colonies of bacteria to compare the amount of bacteria that infiltrated the leaves and thus how sick each genotype became. We were able to graph these results for an easy visual comparison (Fig. 1).

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Fig. 1 Graph comparing the bacterial colonies observed on Columbia-0 and npr1.

This marked the end of our first full-fledged experiment. We used our observations in the UAB Community Gardens to identify possible pathogens by recognizing symptoms of disease (yellow or brown tissue, spots, stunted growth, etc). Next we took samples of infected tissue (admittedly, this part we did in the lab with lab-grade plants after we purposefully infected them) and analyzed the pathogenicity of the bacteria. An important part of a plant pathologist’s job is to determine how different genes are involved in resistance and immune response. We did this by using two genotypes, Columbia-0 andnpr1, and were able to see that the NPR1 gene is involved in resistance–without it, the plants are much more susceptible to disease.

However, OUTPACE isn’t all work and no play. Following the conclusion to our experiment, we played a plant-versus-pathogen board game called Vegevaders. This 2-player game is designed to let users experience the co-evolution between plants and pathogens and understand the basic pathways of pathogenesis and plant immune response. More information, as well as materials for the game, can be found here.

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To make our game more competitive, candy prizes were handed out at the end of the session.image

To bring this week full-circle, we left off by doing another round of infections, except we used a necrotrophic fungal pathogen called Alternaria brassicicola. Unlike our bacterial infections, we did not inoculate the pathogen through the stomatal openings on the undersides of the leaves. Instead we collected spores from fungal cultures grown on V8 plates,

These are the fungal culture plates used to obtain spores for the infection.The top and left plates have already been harvested. The bottom plate is in the process of collecting, and the right plate is waiting to be collected.

Then we counted the spores to make sure the concentration was high enough for an infection,

and pipetted 20μl of spore solution (spores suspended in potato dextrose) directly on top of the leaves. The fungus will be able to infiltrate the leaves on its own by piercing through the cuticle and underlying tissue. We used Columbia-0 as our wild type again, but we used pad2-1 as our susceptible mutant. Next week we will be able to analyze the results from this experiment and compare them to the bacterial infection. It will be interesting to see the differences in how these two pathogens affect the plants and how the two strategies–biotrophic and necrotrophic–vary in terms of pathogenicity and speed of infection.

We collected similar-sized leaves from the plants (Columbia-0 on the left, pad2-1 on the right) and transferred them to a plate for infection.

Close-up on the plate before the leaves are infected.

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