Evaluating Leaf Responses to Microbial Secondary Metabolites Using A High-Throughput Format

My lab works on plant microbe interactions with the ultimate goal of improving seal crop resistance using genetics. Recent developments have shown that microbial secondary metabolites have a key role in promoting plant susceptibility or resistance. To begin, use a cork bore with a four millimeter diameter to collect three leaf discs per plant, while avoiding the mid vein.

With the sharp end apply a twisting motion to prevent macerating the tissue. Add secondary metabolites or solvent treatments at the desired concentration into individual wells of a 96-well plate to reach a final volume of 200 microliters. Gently place the leaf discs into the wells.

Place the 96-well plate with the lid removed into a bell jar equipped with a vacuum nozzle. Then, attach the vacuum hose to the bell jar nozzle and turn on the vacuum pressure at 9.8 pounds per square inch for 10 seconds. Turn off vacuum and slowly release pressure over 10 seconds, and then repeat the vacuum cycle.

Now place the lid back on the 96-well plate and incubate it on an orbital shaker under a light source. Position 23 centimeters above the shaker for four hours. Wash the probe in ultrapure water and dab it lightly on a lint-free wipe to dry.

Note the conductivity reading of the water for future reference. Between four to six hours after treatment, measure and record conductivity in individual wells by immersing the probe into the liquid surrounding the leaf disc. Hold the plate at a 45 degree angle to ensure the probe is fully immersed.

After rinsing the probe is demonstrated earlier, occasionally test the conductivity of the water to confirm probe stability. Then, place a plastic ceiling film over the 96-well plate and return it to the orbital shaker for overnight incubation. To prepare the peroxidase substrate, heat 40 milliliters of distilled sterile water to 70 degrees Celsius in a beaker with a stir bar.

Dissolve 50 milligrams of five amino salicylic acid in heated water. Then, add additional water to bring the total volume to 50 milliliters and adjust the pH to six using sodium hydroxide. Protect the light sensitive solution by covering the container with aluminum foil.

In a 50 milliliter amber conical tube, prepare a 1%hydrogen peroxide solution in ultrapure water. Then, add 10 microliters of the 1%hydrogen peroxide solution per milliliter of five amino salicylic acid solution required to generate the assay medium. At the desired time point, mix five times, and then transfer 50 microliters from each well of the sample plate to a new plate.

Add 50 microliters of the assay medium and incubate at room temperature for three minutes. To stop the reaction, add 20 microliters of two normal sodium hydroxide to each well, and use a plate reader to measure the absorbance of each well at 595 nanometers. Water and 0.1%dimethyl sulfoxide produced similar ion leakage and peroxidase activity levels validating their use as negative controls.

Treatment with one micromolar FLG 22 significantly induced ion leakage, peroxidase activity, and callose production, confirming it as a robust positive control. Gramillin treatment induced significant ion leakage and callose production, but had variable effects on peroxidase activity. Surfactin at 10 micromolar induced both peroxidase activity and callose production while ion leakage remained unchanged.

T-2 toxin treatment suppressed peroxidase activity, while inducing callose production and having no impact on ion leakage. Our hydro assay allows researchers to sample three different stress responses in the same sample, and that enables researchers to make the most of limited amounts of microbial secondary metabolites. The ability to compare 96 samples in the same plate allows researchers to do population genetics level screens, or systems biology in order to understand plant microbe interactions.

My research will continue to focus on plant microbe interactions and characterizing the plant genes that we can leverage in order to develop disease resistance in cereal crops.