A Stepwise Guide to the Isolation and Analysis of Leaf Surface and Apoplastic RNA Using Arabidopsis Rosettes

We are trying to understand how plants regulate their microbiomes and whether RNAs secreted by plants regulate gene expression in both prokaryotic and eukaryotic microbes that colonize plants. We use a combination of high resolution shell electrophoresis and Illumina based analysis to assess the RNAs present on the leaf surface and how they change in response to biotic stress. While recent studies suggest that extracellular RNAs are taken up by fungal and bacterial pathogens, the underlying mechanisms of RNA uptake and functional consequences of this uptake remain largely unexplored.

To begin, take six to seven week old arabidopsis plants for each replicate. Add silhouette L-77 to the sterile VIB, before starting the isolation of leaf surface wash. Then using a measuring cylinder transfer 100 milliliters of the prepared VIB solution into a 500 milliliter spray bottle.

Using fine scissors, carefully detach the arabidopsis rosettes from their roots. After that, use a soft brush to remove any excess soil from the petals. Place the detached rosettes on a flat tray with the abaxial surface facing upwards.

Then spray the abaxial surface using five pumps of the spray bottle filled with VIB and wetting agent. Carefully flip the rosettes over and spray the adaxial surface. Prepare modified syringes with holes at the bottom by wrapping them with para film around the neck to suspend the tip above the bottom of the bottle.

To recover the leaf surface wash, gently insert the sprayed rosettes into the needleless 60 milliliter modified syringes placed inside 250 milliliter centrifuge bottles, and spray two firm pumps of buffer into the syringe. Then centrifuge the loaded bottles at 100 G for 10 minutes at four degrees Celsius. Now filter the recovered leaf surface wash through a 0.22 micrometer syringe filter and collect the filtrate in a 50 milliliter centrifuge tube, kept on ice.

For the leaf surface swab method, spray the detached rosettes with the VIB and wetting agent solution in the same manner as described in the previous step. To recover the leaf surface swab, use sterile cotton tip sticks to swab both the adaxial and abaxial surfaces of the rosettes. Then press the soaked tips against the inner wall of a 15 milliliter centrifuge tube to extract the liquid.

Filter the extracted leaf surface swab sample, through a 0.22 micrometer syringe filter into a two milliliter tube placed on ice. Place the same set of plants previously used for leaf surface wash or swab isolation into a clean beaker and gently rinse them twice with distilled water. To vacuum infiltrate the rosettes with VIB, carefully place the rinsed rosettes into a French press coffee maker containing 300 to 500 milliliters of VIB.

Secure the lid and gently lower the plunger until the rosettes are fully submerged. Insert the French press into a vacuum chamber and apply a vacuum for 20 seconds using a vacuum pump until the plants are well infiltrated. After releasing the vacuum, remove the French press from the chamber and take off the lid.

Pour the VIB into a 500 milliliter plastic beaker and remove the rosettes. Then gently shake the rosettes and brush the leaves across a paper towel to remove excess buffer. Now using blotting paper, gently blot the leaf surfaces to remove any remaining buffer.

To collect the aplastic wash fluid, insert the VIB infiltrated rosettes into 60 milliliter needleless syringes, placed inside 250 milliliter centrifuge bottles as done previously. Centrifuge the bottles at 600 G for 30 minutes at four degrees Celsius with slow acceleration. Then filter the pooled aplastic wash fluid through a 0.22 micrometer syringe filter in a 15 milliliter tube placed on ice.

Prepare or purchase a mini gel containing either 10 or 15%polyacrylamide and seven molar urea in trisboric acid EDTA buffer, using an acrylamide and bis solution. Then mix the RNA samples with two times denaturing loading buffer. Heat the mixture at 65 degrees Celsius for five minutes and immediately place it on ice.

Now resolve the RNA samples in 0.5 times TBE running buffer at room temperature by applying 220 volts for approximately one hour and 15 minutes. Then stain the gel with cyber gold nucleic acid gel stain in 0.5 times TBE for 10 minutes. Rinse the gel twice with distilled water and image it, using an imaging system.

To quantify the resolved RNA bands, use the gel analysis method from the Image J documentation under the analyze and gel analysis menu. Prepare RNA standards for two technical replicates by diluting cell lysate RNA in RNase and DNase free water. To prepare 1.5 times cyber gold nucleic acid gel stain, dissolve it in 500 millimolar tris HCL buffer at pH eight.

For the microplate set up, add 99 microliters of distilled water to each well. Next, add one microliter of RNA to each well using the same volume for both RNA samples and standards. Finally, add 50 microliters of cyber gold solution to each well.

To set the micro plate reader parameters, choose either linear or orbital shaking for three seconds. Set the micro plate reader to fluorescence endpoint mode with an excitation wavelength of 496 nanometers and an emission wavelength of 540 nanometers. Construct a calibration line, using the fluorescence intensity values from the RNA standards.

Denaturing RNA gel analysis revealed that both leaf surface wash and aplastic wash fluid samples contain a broad range of RNA sizes, including both long and small RNA species. The total RNA amounts isolated from the aplastic wash fluid and leaf surface wash were similar, when normalized to plant fresh weight. The RNA profiles of leaf surface wash, aplastic wash, and cell lysate differ markedly, confirming minimal intracellular RNA contamination and showing that aplastic RNA is highly processed with diffuse low molecular weight bands addition of 0.001%silhouette L-77 to the vesicle isolation buffer nearly doubled the RNA yield from leaf surfaces compared to the buffer without the wetting agent.

But increasing this concentration to 0.01%did not further enhance RNA recovery. A second wash of the same leaves recovered approximately 63%of the RNA obtained in the first wash, indicating ongoing RNA secretion onto the leaf surface. RNA quantification, using fluorescence based microtiter plate assays, demonstrated high linearity with RNA concentration for both cyber gold and RIBO 488 detection methods.

We have shown that tRNA and tRNA derived fragments, or TDRs are especially abundant in extracellular RNA, likely due to their resistance to degradation. So we believe that these TDRs likely regulate gene expression in bacteria. We wish to identify the cellular sources of leaf surface RNA and the biological roles this leaf surface RNase might be playing in the context of planned microbe interactions.