酵母の菌タイプIIIエフェクターの発現により誘導される増殖阻害表現型の同定

To begin this protocol, select a vector for the expression of your favorite bacterial effector. In yeast and yeast strain, the vector is then transformed into yeast cells. Next expression of the effector is induced.

Induced cells are lysed for subsequent western blot analysis to verify expression of the effector. After confirming expression of the effector cells are grown and tenfold, serial dilution are spotted onto repressing and inducing media. Finally, after two to three days, yeast growth is analyzed.

Hi, I'm Do Solomon from the laboratory of Dr.Guido Cesa in the Department of Plant Sciences at Tel Aviv University. Today we will show you a procedure for the identification of growth inhibition phenotypes induced by expression of bacterial type three effectors in yeast. We use this procedure in our laboratory to study the function of type three effectors from gram-negative pathogenic bacteria and is a tool that assists us in the identification of the effector TIC targets.

So let's get started. Several important factors should be considered and optimized when designing a yeast system appropriate for expression of the type three effectors of interest. The first is the promoter driving expression of the effectors.

Since bacterial type three effectors may be toxic to yeast cells, an inducible promoter should be used to control their expression. We will use the GAL one promoter in this experiment. The second factor is the copy number of the effector gene.

High expression levels are achieved when the effector gene is carried by a two micron plasmid. Intermediate expression is obtained by using a centromere containing plasmid, and low expression is achieved when the effector gene is integrated into the yeast genome by homologous recombination. A third factor to be considered is the epitope tag used to verify protein expression in cases where antibodies against the effector of interest are not available.

Commonly used tags are M hemagglutinin HA or flag to which reliable commercial antibodies are available. We suggest using only one copy of the tag to minimize undesired deleterious effects on structure and activity of the effector protein. Last but not least, the yeast strain to be used for expression must be tropic to the selectable marker of the expression vector.

To grow yeast cells for transformation, pick a yeast colony from a fresh plate and inoculate three milliliters of YPD in a 15 milliliter polypropylene tube vortex. Briefly place the culture tube in a roller and incubate overnight at 30 degrees Celsius with constant rotation. The next morning, remove the culture from the roller and centrifuge at 800 Gs for five minutes at room temperature.

After centrifugation completely remove the supernatant and resuspend the cells in one milliliter of Resus suspension buffer mixed by vortexing at maximum speed for about 10 seconds. For each plasmid to be transformed and an additional control transformation, transfer 100 microliters of the cell suspension to a micro fuge tube. Add five microliters of single strand salmon sperm, DNA, and 250 to 500 nanograms of plasma DNA to each tube except for the control tube.

Next carefully, add 650 microliters of transformation solution to each tube and vortex. Incubate the tubes at 30 degrees Celsius for 30 minutes on a roller. When the incubation is complete, add 70 microliters of DMSO to each tube and mix by inverting the tubes 10 to 15 times to avoid breaking the cells.

Do not vortex the tubes. Place the tubes in a 42 degree Celsius prewarm water bath and incubate for 15 minutes. After 15 minutes, remove the tubes from the water bath and immediately place them on ice for two minutes.

Once the tubes have cooled down, harvest the cells by centrifugation, then use a pipetter To carefully remove the viscous supernatant. Resuspend each pellet in 150 microliters of sterile double distilled water and spread the cell suspension on a plate with the appropriate selective medium. Leave the plates open for two minutes and allow them to dry in a laminar flow hood.

After that, place the plates in a 30 degree Celsius incubator for two to three days to grow yeast cells. For preparing a protein extract inoculate three milliliters of the appropriate repressing medium with yeast colony from a fresh plate and vortex. Briefly place the culture tube in a roller at 30 degrees Celsius on the following day.

Remove the culture from the roller and centrifuge at 800 Gs for five minutes at room temperature, discard the supernatant and resuspend the cell pellet. In three milliliters of sterile DDW mixed by vortexing centrifuge, again, discard the supernatant and resuspend the cells in three milliliters of sterile DDW. Now transfer 200 microliters of the cell suspension to a new 15 milliliter tube containing three milliliters of the appropriate inducing medium mixed by vortexing.

Place the tube in a roller and incubate overnight at 30 degrees Celsius on the following morning. Transfer one milliliter of the culture or more for low density cultures to a micro centrifuge tube. Harvest the cells by centrifugation at 800 Gs for five minutes at room temperature from here on work in a fume hood to avoid breathing toxic Beamer capto ethanol vapors.

Carefully remove the supernatant using a pipetter and re suspend the cell pellet in 100 microliters of ice cold lysis solution. Mix vigorously by vortexing and incubate on ice for 30 minutes. During the 30 minute incubation, determine the volume of six normal hydrochloric acid required for tittering 100 microliters of lysis buffer to pH nine to 10.

To do this, add various volumes of hydrochloric acid to 100 microliters of lysis buffer in 10 different tubes, and check the pH of the solution using a pH indicator strip at the end of the incubation at the appropriate volume of hydrochloric acid to the lysate and mix by vortexing. Then add 50 microliters of three x sample buffer to the lysate and mix by vortexing puncture a hole in the cover of the micro fuge tube with a needle and boil the lysate solution on heating block for five minutes. After boiling, allow the solution to cool for two minutes at room temperature.

Then vortex the lysate solution and spin it down for 10 seconds. The lysate is now ready for immuno blood analysis to prepare cells for the spotting assay. Grow three milliliter cultures of each strain as shown previously on the following day.

Prepare two plates containing synthetic complete medium without leucine. The medium of one plate should be supplemented with 2%glucose and that of the other plate with 2%actose and 1%raffinose. Dry the plates in a sterile laminar flow hood at room temperature for 20 minutes.

Harvest the cells and resuspend each cell pellet in three milliliters of sterile DDW. Repeat the centrifugation step and resuspend cells again in three milliliters of sterile DDW. Determine the optical density or OD of each culture, and then prepare one milliliter cell suspensions with an OD 600 of one in sterile microfiche tubes.

Next, prepare three tenfold serial dilutions from each of the cell suspensions by using three sterile micro tubes filled with 900 microliters of sterile DDW. Place the dry plates on a grid and for each culture spot, 10 microliters from the four dilution in a row. After spotting, leave the plates open in the hood for several minutes.

When the spots are dry, cover the plates and place them in a 30 degree Celsius incubator for two to three days. Now we will show results from a yeast spotting assay for detection of growth inhibition. Phenotypes induced by expression of type three effectors here, the type three effectors, A-V-R-P-T-O hop, AA one one and AAV RE one of the bacterium.

Pseudomona Riner path of our tomato were expressed from a centromere containing plasmid in a yeast strain on repressing medium yeast strains carrying plasmid for the expression of A-V-R-P-T-O and HOP AA one one exhibited similar growth as the control strain containing an empty vector. While yeast carrying AAV RE one displayed a slightly reduced growth, probably due to leakage of the GAL one promoter and the high cytotoxicity of AV RE one, under inducing conditions expression of either AV E one or HOP AA one one caused a drastic growth inhibition phenotype reflected by the lack of colonies in any dilution. In contrast expression of A-V-R-P-T-O did not exert any growth inhibitory effect.

We've just shown you how to identify growth inhibition phenotypes caused by the expression of bacterial type three effectors in yeast. When doing this procedure, it's important to remember to carefully choose the expression vector as well as this strain, since both can dramatically affect the results. In addition, remember to use freshly transformed these colonies in all steps of the procedure.

So that's it. Thank you for watching and good luck with your experiments.