The overall goal of this procedure is to identify plasmodesmata localization signal in target proteins. The method can help answer key questions with studying plants'intercellular connections by identifying critical thought in signals that direct proteins to the plasmodesmata, which in turn facilitates the cell-to-cell transport of large molecular. The main advantage of this technique is that the procedure is reliable and it can be easily adapted for discovery of plasmodesmata localization signal sequences in most plasmodesmata targeted proteins.
Video demonstration of this method is critical as the planting filtration and preparation for confocal observation steps are difficult to learn by typical print-based publication. Plant Nicotiana benthamiana seeds in wet soil and at a high density. While growing, keep them in a controlled environmental chamber at 20 to 25 degrees Celsius and under 16 hours of light per day, which contains about 75 micromole of photons per meter squared per second.
After the diameter of the u. fil reaches half a centimeter, carefully transfer the seedlings to larger pots and continue their growth in the same chamber under the same conditions. Maintain the plants until they grow to the four to eight leaf stage within four weeks.
At this point, the largest leaves should be four to six centimeters in diameter. For easy identification and analysis, choose an expression system and fluorescently tag each expressed protein as described in the accompanying text protocol. Prepare a pPZP-RCS2-based plasmid as described and add one microgram of it into a 100 microliter culture of competent cells from Agrobacterium tumefaciens.
Incubate the cells on ice for 30 minutes. Then, flash-freeze the cells in liquid nitrogen for one minute. Next, warm the cells at 28 degrees Celsius for 15 minutes and then add one milliliter of LB medium to the competent cell mixture.
After adding the LB medium, place the cells back at 28 degrees Celsius for two hours. Next, spin down the cells at 3, 000 times G for one minute. Resuspend the cell pellet in 0.2 milliliters of LB medium and plate them on antibiotic selective LB agar plates.
Grow the cells on the plates at 28 degrees Celsius for 48 hours until individual colonies are visible. Then, pick and plate several individual colonies onto fresh LB agar plates and grow the cells at 28 degrees Celsius for 48 hours. Next, take a small amount of bacteria from each plate for analysis and use Colony PCR to confirm the presence of the specific binary constructs of interest.
Add each agrobacterium colony with the construct of interest to five milliliters of LB medium supplemented with the appropriate antibiotics. Grow the cells overnight at 28 degrees Celsius. Then, centrifuge the cells at 3, 000 times G.Resuspend the pellet to an OD600 of 0.5 in agroinfiltration buffer.
Incubate the suspension for two hours at room temperature and then load the inoculum into a one milliliter plastic syringe. Holding a fully mature N.Benthamiana leaf with a gloved finger on the adaxial face, press the nozzle of the syringe against the lower epidermis of the leaf. Then, slowly introduce the agrobacterium in infiltration medium by injecting the medium.
Maintain the infiltrated plant until it is to be observed. Use a blade to cut each leaf into fragments between the veins 24 to 48 hours after infiltration. Place the leaf slices on the object slide with the abaxial leaf surface facing up.
Then, place a drop of water on the leaf slice and cover it with the cover glass. Immobilize the cover glass with small pieces of tape on each side. Transfer a slide to under a laser-scanning confocal microscope and use a 10X subjective lens to identify cells with the best signal.
Then, use a 63X subjective lens to record images for more detailed observations. In addition, use the appropriate fluorescents to excite the expressed fluorescent proteins. Retain all of the settings that are used for image acquisition in order to maintain consistency between experiments.
On average, examine 100 to 120 cells for each experimental condition. Diagnose the localization of the tested proteins using the confocal microscope images. These images illustrate the characteristic peripheral punctate plasmodesmata localization patterns observed for the full-length TMV movement protein fused to the cargo molecule CFP, and for the identified plasmodesmata localization signal fused to CFP, as well as for the co-expressed plasmodesmata localization protein, PDCB1 fused to DsRed.
Under control conditions, the plasmodesmata targeting of the full-length TMV movement protein fused to CFP and the localization signal in the TMV movement protein fused to CFP, appears as shown here with sub-cellular localization of CFP and nucleocytoplasmic localization of the marker DsRed2. When the fourth amino acid, valine, is mutated to an alanine, plasmodesmata targeting by both the full-length TMV movement protein and the identified localization signal in TMV movement protein is lost. This indicates that this residue is important for the plasmodesmata localization signal structure or function.
Once mastered, this technique can be finished in 50 hours if it's performed properly. While attempting this procedure, it's important to remember to have the plants in very healthy condition and the plants are at the right leaf stage. After watching this video, you should have a good understanding of, not only how to identify plasmodesmata localization protein signal, but also know how to identify other certain signals in plant proteins.
