This method can help to answer key questions about how to screen the RNA silencing suppressors secreted by plant pathogens. The main advantage of this technique is to provide a rapid, accurate and extensive screening assay for RNA silencing suppressors identification. Prepare potting soil mixes consisting of 50%peat moss, 30%perlite, and 20%vermiculite and autoclave at 120 degrees Celsius for 20 minutes.
Soak the autoclaved soil mixes with plant fertilizer solution at one gram per liter and subpackage them into smaller pots stored in a larger tray. Sow one or two seeds of Nicotiana bethamiana 16c onto the soil surface of each pot. Cover the tray with a plastic dome and allow the seeds to germinate.
Place the tray under light and temperature-controlled growth chambers with a temperature of 23 to 25 degrees Celsius, 50 to 60%relative humidity, and a long day photoperiod. After three to four days, the seeds germinate. Take the plastic dome off and allow the seedlings to grow under the same conditions used for the germination step.
Every two to three days, add an appropriate amount of water keeping the soil moist but not soaking. Every 10 days, add fertilizer to promote further growth. Maintain Nicotiana bethamiana 16c plants under normal conditions until the plants have at least five fully developed true leaves with no visible axillary or flower buds and the leaves have a healthy green appearance.
Use 10 to 14-day-old Nicotiana bethamiana 16c plants for systemic RNA silencing assays and three to four-week-old leaves of Nicotiana bethamiana 16c for local RNA silencing assays. Use a tip to pick a positive colony from the LB plate and inoculate the cells into a glass tube containing five milliliters of LB medium supplemented with 50 micrograms per milliliter Kanamycin and 50 micrograms per milliliter Rifampicin. Grow the cells at 30 degrees Celsius with shaking at 200 RPM for 24 to 48 hours.
Transfer 100 microliters of the culture into five milliliters of LB medium supplemented with the same antibiotics, 10 millimolar MES at pH 5.6, and 20 micromolar AS.Grow the bacteria at 30 degrees Celsius with shaking at 200 RPM for 16 to 20 hours. Centrifuge the cells at 4, 000 times g for 10 minutes. Discard the supernatant and resuspend the pellet in two milliliters of 10 millimolar magnesium chloride buffer.
Repeat the wash to ensure the complete removal of antibiotics. Determine the density of the Agrobacterium culture by measuring the optical density at 600 nanometers. Adjust the cell culture with 10 millimolar magnesium chloride buffer to an OD 600 of 1.5 to 2.0.
Add 10 millimolar MES at pH 5.6 and 150 micromolar AS to the final suspension culture and incubate the cells at room temperature for at least three hours without shaking. Mix equal volumes of an Agrobacterium culture containing 35S green fluorescent protein with an Agrobacterium culture containing 35S cucumber mosaic virus suppressor 2b, putative effector or empty vector. Using a one milliliter needleless syringe, carefully and slowly infiltrate the mixed Agrobacterium suspensions on the abaxial sides of Nicotiana bethamiana 16c leaves.
Remove the remaining bacterial suspension from the leaves with soft tissue wipes and circle the margins of the infiltrated patches with a marker pen. Three to four days post-infiltration, use a long wave ultraviolet lamp to visually detect GFP fluorescence in the infiltrated patches of leaves. Two weeks post-infiltration, use the lamp to detect newly grown leaves of the whole plant for systemic RNA silencing.
To isolate the total RNA from the leaf tissue at four to seven days post-infiltration, collect the leaf tissues from the infiltrated Nicotiana bethamiana 16c patches into a mortar. Place liquid nitrogen into the mortar and grind the tissue into a fine powder with a grinding rod and transfer the powder to a sterile two milliliter tube. Immediately add the RNA isolation reagent at the volume of one milliliter per 100 milligrams of tissue to the sample tube in a hood, vortex vigorously to homogenate, and incubate at room temperature for five minutes.
Add chloroform at the volume of 200 microliters per one milliliter of RNA isolation reagent to each tube in the hood, shake vigorously for 15 seconds, and incubate at room temperature for five minutes. Centrifuge the homogenate at 12, 000 times g for 15 minutes at four degrees Celsius. Transfer the supernatant into a new RNase-free tube and discard the pellet.
Add 0.7 volume of isopropanol to the supernatant, gently invert several times, and incubate the mixture at room temperature for 10 minutes. Precipitate the RNA pellet by centrifugation at 12, 000 times g for 15 minutes at four degrees Celsius. Discard the supernatant.
Wash the pellet with 70%ethanol and air dry the pellet in a hood. Dissolve the RNA in diethyl pyrocarbonate treated water by incubating in a 65 degrees Celsius water bath for 10 to 20 minutes. To perform Northern blot analysis of GFP messenger RNA levels, prepare a 1.2%formaldehyde denaturing agarose gel in 1X MOPS running buffer.
Mix the RNA one-to-one with RNA loading dye and denature the RNA by incubation at 65 degrees Celsius for 10 minutes. Immediately chill the denatured samples on ice for one minute. With a pipette, load the samples into the wells of the gel and electrophorese at 100 volts for 50 minutes until the RNA is well separated.
Rinse the gel into 20X saline sodium citrate buffer to remove the formaldehyde. Perform capillary transfer overnight by setting up 20X saline sodium citrate buffer, one layer of paper towel, two layers of wet Whatman paper, one layer of gel, one layer of nylon membrane, two layers of wet Whatman paper, two layers of dry Whatman paper, one glass plate, and a weight. In the morning, the RNA in the gel is transferred to the nylon membrane.
Soak the membrane in 2X saline sodium citrate and fix the RNA to the membrane by exposing the wet membrane to UV crosslinking. Proceed according to the manuscript. Fully developed leaves of three to four-week-old Nicotiana bethamiana 16c plants were co-infiltrated in patches with Agrobacterium mixtures carrying 35S GFP.
At four days post-infiltration, GFP fluorescence of the infiltrated area was imaged under natural light and long wave UV light. Northern blot revealed that GFP messenger RNA accumulated higher in the leaves expressing 35S GFP plus 35S CMV2b or 35S GFP plus 35S PSR1 than in the leaves expressing 35S GFP plus EV.Agroinfiltration assay was performed to evaluate the spread of the silencing signal in the leaves of two-week-old Nicotiana bethamiana 16c seedlings. At 14 days post-infiltration, more than 98%of the EV exhibited no obvious GFP signaling in systemic leaves.
Whereas both CMV2b and PSR1 efficiently inhibited the systemic spread of the silencing signal. GFP fluorescence was observed in about 80%of co-infiltrated plants and in the remaining 20%of infiltrated plants with only a few red veins appeared in newly emerged leaves. This technique paves the way for researchers to identify novel RNA silencing suppressors secreted by many plant pathogens.
The combination of plants and the proper OD is the most important thing to remember when attempting this procedure. Following this procedure, the functional characterization of the RNA silencing suppressor can be done to explore the mechanism of how these suppressors press the host RNA silencing and what is the output. Please wear the safety goggles when you infiltrate plants and also wear the latex gloves during all the experiment steps.
