Removal and DNA Extraction of Phyllosphere Microbes

This protocol based directly on A Direct Method to Isolate DNA from Phyllosphere Microbial Communities Without Disrupting Leaf Tissues. W Suda, M Oto, S Amachi, H Shinoyama, M Shishido. Microbes Environ. Vol. 23, No. 3, pg. 248-252, 2008. [1] Minor changes were made to the tenses and placements of verbs for protocol clarity.

1. Place 5 g non-shredded fresh leaf sample in a 50 mL sterilized polypropylene tube. Add 5 mL of extraction buffer (100 mM Tris-HCl, pH 9.0, 40 mM EDTA), 1 mL of 10% SDS, and 3 mL of benzyl chloride.
2. Incubate the tube for 15 min at 50°C by mixing repeatedly with 1-min intervals so that the two phases are thoroughly mixed.
3. Remove leaves from the tube, and add 3 mL of 3M sodium acetate (pH5.0).
4. Incubate on ice for 10 min, then centrifuge the suspension (6000xg, 15 min, 4°C).
5. Transfer the aqueous phase to a new centrifuge tube, and precipitate DNA by adding an equal volume of isopropanol followed by centrifugation (9000xg, 15 min, 4°C).
6. Air-dry pellet, then resuspend in 200 uL of TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0).
7. To purify crude DNA extract, use gel filtration. A protocol for gel filtration can be found here ( https://www.nature.com/articles/nmeth0506-411 )

Note to future researchers: Further experimentation is needed to determine if this is actually an effective method of extracting DNA from phyllosphere microbes. Our experiments were not able to sense DNA in the samples extracted from leaves, but the above protocol was only conducted once.

Removal of Live Phyllosphere Microbes for Later Inoculation

1. Attach "nubby" vortex head to the vortexer. Prepare at least 5 TSA plates for later use.
2. Create a 0.01% solution of tween 80 in sterile PBS. As of now, use a working ratio of 5 mL washing solution to 1 g of plant matter.
   1. A very simple way to create this solution is to add 1 uL of Tween80 to each 10 mL of PBS! For example, add 4 uL of Tween80 to 40 mL of sterile PBS. Vortex thoroughly!
3. Using sterile scissors and sterile forceps (sterilized with ethanol OR flame, whichever is the easiest and most effective), hold the individual Arabidopsis by a leaf and snip the stem just above the surface of the soil - transfer to sterile 15 mL conical tube labelled with the sample(s)’ s ID and date obtained.
4. Add 0.01% tween 80 solution to conical tube containing plant matter - use the ratio’s given above! If necessary, close the cap on the conical tube and weigh the tube with plant matter compared to an empty tube, then make calculations.
5. Tape the tube to the nubby vortex head - set the vortexer to around 7 or 8 speed and allow to be shaken for 2 minutes.
6. After vortexing 2 minutes, take 10 uL of original shaken solution and pipet onto pre-marked surface of the TSA plate.
7. Also remove 10 uL of the original solution and dilute 1:10 in sterile water three times to create 10^-1, 10^-2, and 10^-3 aliquots - also pipet 10 uL of each aliquot onto the pre-marked TSA plate surface.
8. Repeat the above steps for the same conical tube for a total of 4, 6, and 8 minutes of shaking.
9. Repeat the above steps with a fresh sample and a fresh conical tube, each time using different TSA plates for each iteration of the experiment.
10. Allow plates to grow at or above room temperature for 2 to 3 days, monitoring at least once a day and recording observations.

Removal of Live Phyllosphere Microbes for Later Inoculation Version 2 (Sonication Method)

1. Create a 0.01% solution of tween 80 in sterile PBS. As of now, use a working ratio of 5 mL washing solution to 1 g of plant matter.
2. Using sterile scissors and sterile forceps (sterilized with ethanol OR flame, whichever is the easiest and most effective), hold the individual Arabidopsis by a leaf and snip the stem just above the surface of the soil - transfer to sterile 15 mL conical tube labelled with the sample(s)’ s ID and date obtained.
3. Add 0.01% tween 80 solution to conical tube containing plant matter - use the ratio’s given above! If necessary, close the cap on the conical tube and weigh the tube with plant matter compared to an empty tube, then make calculations.
4. Place conical tube in sonicating water bath and sonicate for 5 minutes.
5. Use bacterial suspension in conical tube as needed for experiment.

Note to future researchers: The experiments testing the amount of sonication that removed the greatest quantity of morphologically-distinct bacterial CFUs found that 5 minutes was the amount of time best suited for this task. However, further experimentation and an increase in the scale of our initial experiment is necessary to determine if 5 minutes is the actual amount of time to remove the ideal number of microbes from the leaf surface.