The overall goal of the following experiment is to analyze the extent of cell to cell movement of fluorescently tagged protein in plant epidermis. This is achieved by obtaining DNA coated gold particles to express the fusion protein of interest transiently in plant epidermis. As a second step, the DNA is delivered into the plant epidermis, which allows the expression and movement of the tested fusion protein.
Next, the expressed fusion protein is observed under a confocal microscope in order to monitor the expression and localization of fluorescently tagged tested proteins. Results are obtained that show the extent of cell to cell transport of the fluorescently tagged protein based on analysis of the distribution of the fluorescent signal by confocal microscopy. Hi, I'm Ben Myers from the laboratory of KY in the Department of Biochemistry and cell Biology at the State University of New York at Stony Brook.
Today we will show you a procedure for analyzing extent of cell to cell movement or fluorescently tagged protein in plant epidermis. We use this procedure in our laboratory to study cell to cell transport of plant viral movement proteins in various plant backgrounds and to compare the cell to cell mobility of different types of proteins. So let's get started.
To begin this protocol, grow one arabidopsis plant on Promix bx. In an appropriately sized pot, use an environmentally controlled chamber with a short photo period and 40 to 65%relative humidity for nicotiana, hamana and nicotiana tobacco plants grow each plant on Promix BX in a larger pot. Once again, provide an environment controlled chamber with a long photo period and 40 to 65%relative humidity.
Fertilize the plant occasionally with commercially available products as previously described. After growing the Opsis plant for six to eight weeks, select leaves for the experiment that are larger than 15 millimeter by 35 millimeter in size, where the length measurement includes the peole for n benanna and NAC plants grow the plants for seven to 10 weeks. Then select leaves larger than 50 millimeter by 70 millimeter for end benanna and 100 millimeter by 125 millimeter for nac.
In these cases, the length measurements do not include the peole. Before the BIC delivery of DNA coated microparticles can begin prepare the gene gun cartridge with DNA coated gold microparticles as previously described in detail. Then remove the leaves of the same developmental stage with a sharp razor blade, and immediately place them with the AAL sides facing up onto a flat styrofoam surface.
The ABA side of the leaf represents a better substrate for bombardment due to its lower trick home density and thinner cuticle. Small leaves such as a rapid opsis can be immobilized by covering with a piece of window screen mesh and securing the mesh with push pins to the styrofoam surface. Keep the leaves flat to increase the efficiency of the particle delivery and to minimize the damage to the tissue during micro bombardment.
Then insert the cartridge with DNA coated microparticles into the gun. For a rabbit opsis. Perform the shooting at pressures of 140 to 160 PSI for 0.6 micron microparticles.
Bombard the sample using one cartridge per leaf to spread the microparticles over a surface area of 10 to 12 millimeters in diameter. For end benanna and end baum. Perform the shooting at pressures of 160 to 180 PSI for 0.6 micron microparticles with larger end benanna and end tobacco leaves.
Multiple bombardments of the same leaf are possible, however, they must be done at symmetrical positions on each side of the midrib to target the leaf areas at the same developmental stage. The fluorescent signal of the transiently expressed tagged proteins is visualized by confocal. Microscopy care should be taken to find optimal microscopy settings for detection of each tested protein.
Use a 40 x objective lens to observe proteins that show limited intracellular accumulation with weak signal intensities, such as the plasma desal localization of the tobacco mosaic virus, movement protein or T-M-V-M-P. This affords higher resolution and enhanced sensitivity. However, a 10 x objective lens with a confocal zoom function can be used for faster imaging of proteins that show cytoplasmic distribution with strong signal intensities such as free yellow fluorescent protein or YFP Simplistic transport is inferred from the appearance of multi-cell clusters that contain the fluorescent signal.
The number of such clusters and the number of cells in each cluster is indicative of the extent of cell to cell transport. To obtain reliable data record at least 100 expression. Clusters per experimental system, take care to carry out experiments simultaneously.
When comparisons will be made. Here, the simplistic transport of fluorescently tagged proteins was detected. Typical confocal images that are obtained following micro bombardment of an end hamana leaf with A TMV mp YFP expressing construct are shown.
Simplistic movement of TMV MP YFP is observed based on the appearance of multi-cell clusters of the YFP signal. However, not all transiently expressed TMV MP YFP is able to move as evidenced by single cell signal. In some micro bombardments.
Statistically in less than 40%of the counted signal clusters, TMV MP YFP is unable to move between cells. Whereas in greater than 60%of the clusters, the protein moves between two and five cells. With the two cell spread being the most frequent, the proteins with relatively small molecular size without innate movement activity can diffuse through plasma SMA to move cell to cell.
For example, free YFP or one XYFP spreads between several cells and greater than 30%of the counted clusters. Conversely, this non-specific diffusion does not occur for a translational YFP dimer or two XYFP, which is comparable in size to TMV MP YFP fusion protein and is completely cell autonomous. We've just shown you how to analyze cell to cell movement of fluorescently tagged proteins using transient expression by a gene gun system in plant epidermis.
When doing this procedure, it's important to remember to prepare healthy, robust plants, high quality DNA coated gold particle for delivery of DNA and to analyze enough expression clusters to obtain statistically reliable results. So that's it. Thanks for watching and good luck with your experiments.
