The overall goal of the following experiment is to monitor the interaction of two proteins expressed in intact tobacco leaves. This is achieved by designing appropriate constructs, fusing the two genes of interest to split fluorescent proteins and transforming these constructs into agro bacteria. As a second step agro bacteria cultures are mixed and injected into tobacco leaves, which leads to expression of the proteins and a reconstituted fluorescent signal.
If the proteins come into close proximity, next, either entire leaves or isolated protoplasts are analyzed under the microscope. Results are obtained that show protein protein interactions based on the emitted fluorescent signal detected by fluorescence microscopy. The main advantage of this technique of existing methods like cor immunoprecipitation or yeast to hybrid is that the protein protein interaction can be directly monitored in the living plant cell.
This method can help to answer key questions in the plant biology field, such as the formation of protein complexes in various cellular compartments. To begin this procedure, grow the agro bacteria to be used for transformation of the tobacco leaves. Inoculate 10 milliliters of LB medium containing the appropriate antibiotics with 50 microliters of the AG L one glycerol stock culture containing the plasmid of interest in a sterile 50 milliliter tube.
Incubate at 28 degrees Celsius for at least 24 hours, shaking at 190 RPM until the culture reaches an OD 600 between 1.0 and 2.0 on the following day. Centrifuge the bacteria at 3000 times G for 15 minutes. After discarding the supernatant resus, suspend the pellet in freshly made infiltration medium and adjust the suspension to an OD 600 of 1.0.
Incubate the agro bacteria cells in an overhead shaker for two hours in the dark at room temperature for infiltration of tobacco leaves. Choose several older leaves from a three week old tobacco plant. Mix equal volumes.
Three milliliters each of the agro bacteria carrying the constructs of interest. Fill a five milliliter syringe without a needle with the cell suspension mixture to infiltrate the cell suspension into the tobacco leaves. Carefully press the syringe on the bottom side of the leaves in several places, water the plants and leave them covered and protected from light for two days.
To isolate protoplasts from an infiltrated leaf, first, place the leaf into a Petri dish and add some freshly prepared enzyme solution. Using a new razor blade, cut the leaf into approximately 0.5 square centimeter size pieces. Next, transfer the leaf pieces with the enzyme solution into a vacuum.
Infiltration flask. Vacuum infiltrate for approximately 20 seconds until air bubbles emerge from the leaves. Release the vacuum very carefully.
Shake the flask for 90 minutes at 40 RPM in the dark at room temperature after 90 minutes. Release protoplasts by shaking for one minute at 90 RPM. Filter the solution through gauze into a 15 milliliter round bottom centrifuge tube.
Overlay the protoplasts solution with two milliliters of FPCN buffer and centrifuge for 10 minutes at 70 times G with slow acceleration and deceleration at room temperature. Intact Protoplasts will accumulate at the interface between the enzyme solution and FPCN using a wide orifice one milliliter pipette tip transfer the intact protoplasts into a fresh centrifuge tube. For success of this procedure, always use white orifice tips to prevent rupturing of intact protoplasts.
Fill up the tube with W five buffer centrifuge for two minutes at 100 times G with slow acceleration and deceleration. To pellet the protoplasts, remove the supernatant carefully and resuspend the pellet. In approximately 200 microliters of W five buffer, depending on the amount of protoplasts.
To prepare a protoplasts sample for laser scanning microscopy first pace, two small strips of sealant about two centimeters apart on a microscope slide. Place 20 microliters of the protoplasts solution between the strips and carefully place a cover glass on top. The sealant strips will prevent the protoplasts from being squashed by the cover glass.
To prepare a total leaf sample for laser scanning microscopy, cut a one centimeter piece from the leaf and place it onto a microscope slide with the bottom side of the leaf facing upwards. Add approximately 30 microliters of water. Place a cover glass on top and fix it tightly with adhesive tape on both sides.
Imaging is performed with a confocal laser scanning microscope from MICCA type TCS SP five for magnification. Use an objective lens with a magnitude of 63 x with glycerol as the imaging medium, use the Leica application suite advanced fluorescent software for evaluation. Set the Argonne laser to 30%and the laser power at 488 nanometers to an intensity of 18%To monitor it signal at 515 nanometers, set the first PMT detector emission bandwidth from 495 to 550 nanometers.
To monitor chlorophyll autofluorescence. Set a second PMT detector emission bandwidth from 650 to 705 nanometers. To monitor M cherry signal, use the HENI 5 61 laser.
Set the intensity of the laser to 18%and a third PMT detector emission bandwidth from 587 to 610 nanometers. Make sure that pictures from all PMT detector channels are taken with the same gain settings. The gain should be between 800 and 900 to exclude background signals.
Acquire images in this format, width and height of 1024 by 1024 pixels with a scan speed of 100 hertz. For Z stackings, use a maximum distance of 0.5 microns between each stack. In this study, the BFC method was used to monitor the interaction of the cytosolic molecular chaperone HSP 90 with the membrane docking proteins TPR seven and to 64.
As shown in this schematic, Venus is coupled to the cytosolic part of either TPR seven, which resides at the endoplasmic reticulum or to 64, which resides in the chloroplast outer envelope. Hs.P 90 is N terminally fused to SCFP enabling interaction of the TPR domains of talk 64 and TPR seven With the HSP 90 C, Terminus, SCFP alone is expressed in the cytosol as a negative control to verify the localization of the TPR seven HS P 90 protein complex. TPR seven and HS P 90 were co transformed with an ER marker.
The fluorescence was monitored in intact leaves as a control. SCFP alone was expressed along with TPR seven and the ER marker. In all images shown, the scale bar represents 10 microns.
The panels on the left show reconstituted fluorescence in green monitored at 515 nanometers. The ER marker appears in red. In the middle panels overlay of both signals is shown in the right panels.
A reconstituted signal for TPR seven together with HS P 90 was monitored overlapping with the ER marker appearing in yellow. In contrast, no signal for TPR seven and the negative control as CFP was monitored. In the next example, the chloroplast protein tox 64 was co-expressed with HS P 90 as a control.
Tox 64 was co-expressed with SCFP alone. As before, reconstituted fluorescence in green was monitored at 515 nanometers. The middle panels show chlorophyll autofluorescence monitored at 480 nanometers.
In red overlay of both signals is shown in the right panels. Reconstituted fluorescence was observed in cells coex expressing tox 64 and HSP 90, but not in cells. Coex expressing tox 64 and SCFP alone.
Since the exact localization of tox 64 and HSP 90 is difficult to determine in microscopic pictures of the entire leaves. Protoplasts were isolated from infiltrated tobacco leaves for fluorescence microscopy. As before, reconstituted fluorescence was monitored at 515 nanometers chlorophyll autofluorescence was monitored at 480 nanometers and overlay images were created as shown in this overlay image to 64 and HSP 90 can be detected as ring shaped structures surrounding the chloroplast Off.
After watching this video, you should have a good understanding how to design your constructs, transform tobacco leaves, and how to best visualize the fluorescent signal showing the interaction of your two proteins of interest.
