The overall goal of this procedure is to unravel virulent strategies and host pathogen systems by identifying the interaction of a candidatus phytoplasma malefactor protein with host proteins from malus domestica. This method can help answer key questions in many different biological research fields, and is here used in plant research to elucidate molecular mechanisms underlying the development of Apple Proliferation Disease. The main advantage of this technique is that a single pathogen effector can be screened against thousands of potential interactors, making this method an easily feasible starting point in infection research.
To begin, identify infected trees with Apple Proliferation specific symptoms and control trees that are symptom free. Using clean secateurs, cut root samples from three different sites of the root system that have a diameter of 0.5 1 cm, and a length of about 5 cm. Place the root samples into adequately labeled plastic bags.
Then place the samples in a cold box with chilled thermal packs and store them at four degrees Celsius until further processing. Rinse the root samples with water to remove the soil. Then transfer the samples into a sterile Petri dish and use a sterilized scalpel to remove the root epidermis and the cortex.
With a clean, lint-free tissue, wipe the scalpel. Then dip the instrument in 70%ethanol and heat sterilize it over an open flame. Use the scalpel to scratch the phloem.
Chop the sample into small pieces and aliquot 30-100 milligrams of the pieces into a sterile two milliliter reaction tube. Store the samples at 80 degrees Celsius or use them immediately to isolate DNA, which serves as template for effector gene amplification and subsequent cloning of the effector into the bait vector. After isolating phytoplasma polyspecific DNA from infected trees, cloning into bait vectors and testing it for self-activation and expression according to the text protocol.
Streak plex A ATP 00189 effector transformed NMY51 onto a fresh SD-trp plate and culture it at 30 degrees Celsius for two to three days until red colonies appear. Use a red colony from the agar plate to inoculate three milliliters of SD-trp medium in a small shaking flask and incubate the culture overnight at 30 degrees Celsius with shaking at 120-150 rotations per minute. The following day, with one milliliter of the overnight culture, inoculate 20 milliliters of SD-trp in a shaking flask and let it grow for eight hours.
Use SD-trp medium to adjust the culture to an OD600 of 0.2, and with ten milliliters of the culture, inoculate two flasks containing 100 milliliters each of SD-trp medium. Incubate the cultures at 30 degrees Celsius with shaking overnight. Next, measure the OD600 of the culture and pellet 120 OD600 units.
For example, if an OD600 of 1.2 is measured, spin down 100 milliliters of sample, discard the supernatant, and use 800 milliliters of pre-warmed 2xYPAD to resuspend the pellet into two shaker flasks and incubate at 30 degrees Celsius. Incubate the yeast culture at 30 degrees Celsius and 120-150 rotations per minute. Measure the OD600 about every 1.5 hours according to the text protocol until an OD600 of 0.6 is reached.
After preparing salmon sperm DNA, Te Lithium OAc and PEG Lithium OAc according to the text protocol, centrifuge 800 milliliters of yeast culture at 700 x G for five minutes to pellet the cells. Remove the supernatant and resuspend the pellets in a total of 200 milliliters of sterile double-distilled water. Then pellet the cells again and discard the supernatant.
Resuspend the pellet in 16 milliliters of Te Lithium OAc mix and spin down the sample again. Then after discarding the supernatant, use 9.6 milliliters of Te Lithium OAc to resuspend the pellet. In appropriately sized reaction vessels, prepare twelve vials with seven micrograms of peak Add HAC DNA library vector, 100 microliters of 2%salmon sperm DNA, and 2.5 milliliters of PEG Lithium OAc mix.
Add 600 microliters of the previously prepared yeast cell suspension to each of the twelve vials and mix vigorously for one minute. Then incubate the reactions in a water bath at 30 degrees Celsius for 45 minutes mixing every 15 minutes. Next, add 160 microliters of DMSO to every vial and mix vigorously.
Then incubate the vials at 42 degrees Celsius for an additional 20 minutes. After pelleting the cells, discard the supernatant and use three milliliters of 2xYPAD to resuspend each pellet. Pool the cells from all twelve vials in a 100 milliliter shaker flask and incubate the yeast for 90 minutes at 30 degrees Celsius and 120 rotations per minute.
Following the incubation, after pelleting the cells and discarding the supernatant, use a ten milliliter serological pipette and 4.5 milliliters of sterile 0.9%sodium chloride to thoroughly resuspend the pellet by carefully pipetting up and down. Withdrawal 50 microliters of the suspension and use 0.9%sodium chloride to prepare tenfold dilutions from 1:10 up to 1:1000. Then plate 100 microliters of each dilution on 90 millimeter Petri dishes containing SD-trp-leu agar.
Spread the rest of the undiluted yeast suspension on 16x150 millimeter diameter Petri dishes with SD-trp-leu-His-ade agar. Incubate the plates at 30 degrees Celsius for three days for the SD-trp-leu plates and for four days for the SD-trp-leu-his-ade plates. Determine the transfection efficiency by counting the colonies of the different serial dilutions on the SD-trp-leu selection plates.
Transfer each clone by using a sterile pipette tip to pick and streak the colony onto fresh SD-trp-leu-his-ade selective plates. Incubate the plates at 30 degrees Celsius for 24 hours. Repeat the transferring of clones every day until a total of five passages is reached.
To analyze clones, under a sterile hood, prepare one sterile two milliliter reaction tube with one milliliter of SD-trp-leu-his-ade for each clone. Use a hot needle to punch a hole into each tube and use a gas permeable sealer to cover the hole. Inoculate each vial with fresh colony material from one clone and incubate the vials at 30 degrees Celsius and 150 rotations per minute for 24 hours.
After pelleting the cells and discarding the supernatant, resuspend the pellet in the appropriate buffer and transfer it to a fresh two milliliter reaction tube. Add 100 microliters of acid washed glass beads to the suspension and mix the tube vigorously for five minutes. Finally, purify the plasmid DNA according to the text protocol.
A summary of expected results of the self-activation assay and their interpretation is provided in this table and figure. Weak self-activation leads to growth on trp-leu-his, but not on trp-leu-his-ade depleted selection plates. While yeast transformed with a strong self-activating bait would grow on trp-leu-his-ade lacking medium.
A successful cotransformation of bait and prey is characterized by growth on selective plates lacking trp and leu. Interaction of the bait and a prey protein leads to a complementation of the his and ade oxytrophy of NMY51 Shown here is an example of an interaction between bait and prey in a Yeast 2 Hybrid Experiment. The malus domestica host interaction partners, MdTCP24 and MdTCP25 were identified and interaction was confirmed by denovo cone transformation with the effector.
Once mastered, the yeast 2 hybrid can be performed in one day if all necessary equipment and materials, especially the yeast, is properly prepared in advance. While attempting this procedure, it is important to avoid contamination and always work under sterile conditions. Following this procedure, another independent method must be performed like bimolecular fluorescence complementation for example, to confirm the found protein-protein interactions.
This is particularly important since the yeast or hybrid per se is relatively prone for generating false positive results. After it's development, this technique paved the way for researchers in many different research fields to better understand the molecular principles that involved protein-protein interactions, especially in host-pathogen interactions, and many other biological research fields. Furthermore, you will understand that performing this method is easily feasible in any decently equipped molecular biology lab.
Don't forget that working with certain chemicals, scalpels, and open flames can be extremely hazardous, thus when performing this procedure, you always have to consider certain precautions. That means use your personal safety equipment like gloves and goggles, and use the general laboratory safety equipment.
