Bacterial Leaf Infiltration Assay for Fine Characterization of Plant Defense Responses using the Arabidopsis thaliana-Pseudomonas syringae Pathosystem

The overall goal of this procedure is to quantitatively characterize Arabidopsis diliana immune response through an optimized syringe infiltration assay. This is accomplished by first using syringe infiltration to manually deliver bacteria into the leaf through the natural openings called sada. The second step is to obtain discs of infected leaf tissue after the development of disease symptoms.

Next, the bacteria are extracted from the leaf discs through high throughput homogenization. The final step is the serial dilution and spotting onto bacterial counting plates to enumerate colonies. Ultimately, bacterial growth within each leaf diss can be correlated to the relative strength of the plant immune response.

This method can help answer key questions in the field of plant immunity, such as identifying genes that contribute to defense responses against biotic stress. The main advantage of this technique over existing method is that it allows precise quantification of pathogen growth in the infected plant tissue and helps avoid experimental error caused by sampling leaves with varied developmental status. This method can provide insights into the plant immune response to an infection with a virulent bacterial pathogen, and it can also be applied to other systems such as salicylic acid or microbe associated molecular patterns triggered immune responses.

Generally, individuals new to this method will struggle with delivering the bacteria into the leaf without tissue damage. Demonstrating the following procedure will be postdoctoral scholar Hindu and graduate students shalu and yali. Soon from my laboratory, The assay for enhanced disease susceptibility will be demonstrated in this video.

Prior to the experiment, prepare King's B or KB media plates make one liter of KB solid medium as described in the protocol text and let it cool on a magnetic stir plate. When the media is cooled to touch at 80%glycerol one molar magnesium sulfate and streptomycin to the media pour two different sizes of KB media plates. The smaller Petri dish serves as the primary bacteria culture plate and the larger pet Petri dish serves as the bacteria counting plate.

Store the plates at four degrees Celsius two days before the assay streak. PSM ES 43 26. Bacteria from a negative 80 degree Celsius glycerol stock on a KB strep bacteria culture plate and incubate at 28 degrees Celsius for 24 to 48 hours on the following day.

Initiate the liquid culture in a sterile test tube with four milliliters of liquid kb medium containing 50 micrograms per milliliter of streptomycin. Shake a 250 RPM at 28 degrees Celsius overnight. The Arabidopsis plants for the experiment are grown under conditions detailed in the protocol text on the same day that the liquid bacterial culture is initiated.

Mark the petals of leaves number five and six with a blunt and waterproof marker for easy identification of infected tissue at sampling. To enhance the opening of STA and facilitate the entrance of pathogen solution into the leaf water. The plants well by soaking the flat from the bottom for 20 minutes.

After 20 minutes, drain the excess water. The infection of the arabidopsis plants is carried out. The day after initiation of the liquid culture.

Transfer the bacterial culture to a 1.5 milliliter micro centrifuge tube and spin at 9, 600 Gs for two minutes. At room temperature, discard the supernatant and resuspend the bacterial pellet with one milliliter of sterile 10 millimolar Magnesium chloride dilute the bacteria suspension with nine milliliters of 10 millimolar magnesium chloride in a 50 milliliter centrifuge tube. After measuring the optical density of the bacteria, dilute the bacteria with 10 millimolar magnesium chloride to a final OD 600 of 0.0002.

For the EDS infection assay. A one milliliter blunt end needleless syringe, commonly known as an insulin syringe is used for infiltration. Fill the syringe with 0.5 to 0.6 milliliters of the diluted bacterial solution for much better control during the infiltration process, do not fill the syringe to its full capacity.

Expose the lower surface of the leaf on the top of the index finger, and then with the help of the thumb, gently adjust the leaf position. Position the syringe vertically against the leaf surface to ensure even distribution of pressure to reduce leaf damage. Try to avoid the midrib area during the infiltration.

Slowly push the plunger to infiltrate the bacterial solution. Liquid entry into the leaf mesophyll will be visualized as indicated by the darker leaf color, infiltrate the entire surface of the leaf even if it requires multiple attempts. Once infiltration is complete, gently blot the leaf with absorbent tissue to remove the extra pathogen solution.

Visually inspect the infected leaf tissue for damage. Leave the infiltrated plants to dry for one to two hours, return the plants to their original growth conditions. Spray a clear dome with water and cover the infected plants for two hours.

Then crack the dome to generate a two to three inch opening and leave it on throughout the remainder of the infection Experiment, which is usually three days. One day before quantifying pathogen growth. Pred dry the 150 millimeter by 15 millimeter KB media plates.

Dry the plates by keeping them at room temperature for about 24 hours. Process the infected tissue after the emergence of sclerosis indicated by the yellowing of the infected tissue in the susceptible genotypes, but before the development of necrotic lesions. For each genotype, prepare six grinding tubes.

Place one stainless steel grinding ball and add 500 microliters of sterile, 10 millimolar magnesium chloride into each tube. Next, detach the infected leaf from the plant and punch a leaf disc with a one hole paper punch using forceps randomly, place two leaf discs from two different plants into each grinding tube. Seal the tubes, homogenize the tissue with a high throughput homogenizer at maximum speed for 10 minutes.

Repeat this process if needed until the tissue is well homogenized and the solutions turn green due to chlorophyll release from the infected leaves while waiting. For the homogenization, fill each well in the first six rows of a 96 well culture plate with 180 microliters of 10 millimolar magnesium chloride. Transfer 20 microliters of the ground tissue suspension into each well of the first row of the 96 well plate and mix by repeatedly pipetting the liquid up and down.

If small fragments of tissue clogged the tip, clip it by two to three millimeters to help acquire the correct volume of the solution to provide enough space for the droplet on the top of the plate space the tissue from different genotypes in alternative rows. To prepare a tenfold serial dilution transfer 20 microliters of tissue suspension into the second row and repeat this procedure until the sixth dilution. Using divided pipette tips, transfer 20 microliters of the solution from the 96 wall plate onto the 150 millimeter by 15 millimeter KB plate.

Work from the most dilute suspension to the most concentrated so it is unnecessary to change pipette tips between the dilution. Dry the plate at room temperature with the lid cracked. Once no more liquid can be observed on the plate surface, close the lid invert and incubate the plate at room temperature.

Incubate plates for 40 to 60 hours until the colonies become visible. Confirm that the bacteria on the plates reflects the predictable tenfold. Drop in colony forming units.

Count the bacteria before they overgrow and colonies fuse. Determine the number of bacteria in the lowest dilution that does not have overlapping colonies. Usually the preferred dilution to be counted will contain between 10 and 50 colonies, and it may vary among technical replicates.

To calculate the levels of bacterial proliferation, determine the number of colony forming unit per leaf disc using this formula where R is the number of the row and T is the number of the bacteria within each technical replicate. The data is then used to produce a graph. Each data point is represented as the mean of six technical replicates on a logarithmic scale.

Aerobars represent the 95%confidence interval of the mean enhanced disease. Susceptibility against P-S-M-E-S 43 26 was assessed by infection with an inoculum with an OD 600 of 0.0002. The results show that the highly susceptible NPR one dash one mutants have approximately 2.5 log or 300 times more pathogen growth compared to the wild type Columbia zero plants.

The syringe infiltration assay can be modified to assess different layers of immune response. To evaluate salicylic acid triggered immunity, external application of sodium salicylate is used to trigger the immune response, which is quantified by pathogen growth. The loss of NPR R one, which functions as the salicylic acid receptor and major transcriptional co-regulation of salicylic acid dependent target genes leads to insensitivity to exogenous application of the salicylic acid derivative.

To characterize microbe associated molecular pattern triggered immunity, plants were pretreated with two known triggers. FLAG 22 and L 18, the FLAG 22 treated wild type Columbia zero plants supported a one log or 10 times reduction in the bacterial population while the FLS two mutant plants failed to trigger the bacterial growth restriction effect. Similarly, the EFR mutant plants were insensitive to L 18 pretreatment.

After watching this video, you should have a good understanding of how to quantify the Arabic opsys immune response through the syringe infiltration assay. While attempting this procedure, it is important to remember to monitor the disease development and sample the tissue after the emergence of corsis. The pseudomona pather cul cola S phos 3, 2 6 is a hemi atrophic pathogen.

Thus, the sampling should be conducted before the transition into the neurotrophic stage. Following this procedure, other methods like quantitative real time PCR and Western blood analysis can be performed in order to answer additional questions like which particular immune signaling pathway is disturbed. Don't forget you're working with living bacteria.

Although Pseudomona Senge is not pathogenic on humans, please remember to properly dispose of contaminated consumables and infected plants while performing this procedure.