Hi, I am Suma TI from the lab of Greg Martin at the Voice Thompson Institute for Plant Research. Today we will show you a procedure for a cell death based assay for PAM triggered immunity or PTI. We use this procedure in our lab to look for the involvement of a gene in PTI.
So let's get started. Begin the procedure by growing Nicotiana ANA until they're about seven weeks old. Four to five days prior to starting.
The PAM triggered immunity or PTI assay. Trim the plants to remove all axillary branches and flowers. Try removing the axillary branches soon after they emerge to make the plants more manageable.
After preparing the plants proceed to grow the bacteria. This procedure uses non-pathogenic bacteria for induction of plant immunity prior to a second inoculation with challenge bacteria, initiate growth of all the bacteria used in the assay. At the same time, use frozen glycerol stalks of the pseudomonas bacteria for each pseudomonas bacterial stalk.
Spread a small amount of bacteria on the center of A KBM plate containing the appropriate antibiotic and incubate the plate at 30 degrees Celsius for about 18 to 24 hours. For the agrobacterium, use a previously prepared fresh plate to start a liquid culture in two milliliters of lb. Grow overnight at 30 degrees Celsius with shaking on the following day.
Add 150 to 200 microliters of liquid KBM to the plates of the pseudomonas bacteria. Spread the bacteria over the entire plate. Using a sterile greter.
Put the plates back in the incubator for another 20 to 24 hours. Four agrobacterium. Start a secondary culture in about five to 10 milliliters of LB containing 20 micromolar acetone.
Grow the culture overnight at 30 degrees Celsius with shaking. After preparing the bacteria proceed to prepare the plants for the assay the day before conducting the PTI assay. After preparing the bacteria, move the plants to a 22 to 24 degrees Celsius room with approximately 35 to 40%relative humidity and constant light.
Next, use a thick black marker to mark circles on the leaves. Choose well expanded leaves and avoid older leaves or leaves that are tough or thick to the touch. Avoid marking the lower portions of the leaf.
The circles are at least 1.5 centimeters in diameter and two to four circles can be marked per leaf. The circles should be well spaced and preferably not cross a large vein. Start the PTI assay by harvesting the bacteria used in the induction of PTI.
The pseudomonas species used should have formed a bacterial lawn indicative of good growth. Harvest them from the plate by scraping off with a sterile long wooden stick and resuspend in about 20 to 25 milliliters of 10 millimolar. Magnesium chloride solution for agrobacterium centrifuge.
The culture to collect the cells and resus suspend in about 20 to 25 milliliters of 10 millimolar magnesium chloride plus 10 millimolar. MES pH 5.6, repeat the centrifugation and again, resus Suspend in about 20 to 25 milliliters of the magnesium chloride plus MES solution. Now that the inducing pseudomonas and agrobacterium bacteria are resuspended.
Measure the optical density of all three cultures at a wavelength of 600 nanometers. Adjust the ODS by diluting the cells with magnesium chloride for the pseudomonas and MES plus magnesium chloride for the agrobacterium. The final required OD values are found in the accompanying written protocol.
A total volume of 25 milliliters of bacterial culture is sufficient to inoculate about 40 to 50 spots. Now start the assay by infiltrating the PTI inducers into the pre-marked circles on the leaves using a one milliliter syringe without a needle. Write down the order in which the plants were infiltrated and the exact time when the infiltration was begun following the induction.
Proceed to the challenge inoculation several hours after the induction inoculation. Harvest the bacterial strains required for the challenge inoculation as previously shown for the inducing pseudomonas strains. Again, measure the OD and adjust the concentration of the cultures according to the accompanying GR protocol.
Start the challenge inoculation seven hours after the inducer infiltration. Use the following inducer challenger combinations. Choose a point on the periphery of the first inoculation circle as the center of the second inoculation circle.
Perform the challenge infiltration in the same order of plants as that of the induction. If there are multiple spots on a leaf, then make sure the infiltrations do not overlap. Finally, plate serial dilution of all the cultures used in the assay on KBM or LB plates containing the appropriate antibiotics.
Two days after the challenge inoculation one can start evaluating the PTI response two days after the induction and challenge inoculations look for the appearance of cell death due to effector triggered immunity or ETI in the spots that were challenged with PST DC 3000 cell death inside the overlapping area of infiltration indicates a breakdown of PTI and should be scored as a positive phenotype four to five days after the induction and challenge inoculations look for the appearance of cell death due to disease in the spots that were challenged with the DC 3000 hop Q1 mutant or PS back high again. Look for breakdown of PTI that is indicated by cell death due to disease in the overlapping area of infiltration. Continue to observe the plants until those which are not compromised for PTI begin to show cell death in the overlapping area of infiltration.
Plants silenced for FLS two are compromised for PTI two days after inoculation with the PFDC combination. Cell death is seen in the overlapping area indicating breakdown of PTI control plants that have not been silenced for any gene. Were also inoculated with the PFDC combination and examined two days after conducting the assay cell death due to the challenge is seen outside but not inside the overlapping area due to a normal PTI response.
We've just shown you how to perform a cell death based assay for pant triggered immunity or PTI while doing this procedure. It's important to remember to keep the plants in the environmental conditions that we recommend. Higher levels of humidity would negatively affect the plants and make the assay proceed more quickly.
So that's it. Thanks for watching and good luck with your experiments.
