The traditional approach to analyzing the function of microRNAs in plants primarily relies on the generation of stable knockdown plants. This virus-based microRNA silencing system does not require tedious and time-consuming transformation. Virus-based microRNA silencing is a rapid and efficient tool for functional characterization of microRNAs in plants that can be applied to a broad range of diploid and tetraploid potato species.
Demonstrating the procedure will be Jinping Zhao, a post-doctoral research associate from my laboratory. To design the target mimic module, insert a mismatch sequence into the reverse complement sequence of the microRNA of interest at the site corresponding to the 10th to 11th nucleotides of the microRNA. To amplify the short tandem target mimic fragment, set up a PCR reaction with each primer of interest, a 48 nucleotide space oligo, 10 times PCR buffer, a dNTP mixture, high fidelity PCR DNA polymerase, and double distilled water to a final volume of 50 microliters.
Then amplify the microRNA by standard PCR using the parameters as indicated. To purify the short tandem target mimics, add 2.5 volumes of ethanol and a 1/10th volume of three molar sodium acetate to the PCR product. Mix vigorously before centrifuging the reaction at 14, 000 times G for 10 minutes.
After removing the supernatant, rinse the pellet with one milliliter of 70%ethanol and allow the pellet to dry, then dissolve the pellet in 20 microliters of double distilled water. To set up a T4 DNA polymerase reaction, mix the purified short tandem target mimic PCR product with 10 times T4 DNA polymerase buffer, one molar dithiothreitol, 100 millimolar dATP, T4 DNA polymerase, and double distilled water to a final volume of five microliters on ice. Then incubate the mixture for 15 minutes at 37 degrees Celsius, followed by a 20-minute incubation at 75 degrees Celsius to inactivate the T4 DNA polymerase.
To prepare the PVX-based VBMS construct, digest five micrograms of PVX ligation independent cloning plasmids with 2.5 microliters of SMA1 in a total volume of 100 microliters. Add an equal volume of phenol to chloroform to isoamyl alcohol, add a 25 to 24 to one ratio to the digested PVX ligation independent cloning products with vigorous mixing and collect the product by centrifugation. Add an equal volume of chloroform to isoamyl alcohol at a 24 to one ratio to the supernatant with vortexing before subjecting the tube contents to a second centrifugation.
At the end of the spin, transfer the supernatant to a new centrifuge tube and add 2.5 volumes of ethanol and a 1/10th volume of three molar sodium acetate with vigorous mixing. Centrifuge the tube and rinse the pellet with one milliliter of 70%ethanol and vigorous vortexing. Centrifuge the tube again and remove the supernatant to allow the pellet to air dry.
Then dissolve the digested PVX ligation independent cloning plasmid pellet in 100 microliters of double distilled water. To set up the T4 DNA polymerase reaction, mix the digested PVX ligation independent cloning vector DNA with 10 times T4 DNA polymerase buffer, one molar dithiothreitol, 100 millimolar dTTP, and T4 DNA polymerase to a final volume of five microliters on ice. Then incubate the mixture at 37 degrees Celsius for 15 minutes, followed by a 20-minute incubation at 75 degrees Celsius to inactivate the T4 DNA polymerase.
To clone the short tandem target mimic sequence into the PVX ligation independent cloning vector, mix five microliters of the T4 DNA polymerase-treated short tandem target mimic PCR product with five microliters of the T4 DNA polymerase-treated PVX ligation independent cloning plasmid and incubate the mixture for five minutes at 70 degrees Celsius. At the end of the incubation, cool the tube contents to 22 degrees Celsius in a PCR machine at a rate of 0.1 degrees Celsius per second, leaving the tube in the thermocycler for 30 minutes once the target temperature is reached. At the end of the incubation, transform five microliters of ligation independent cloning reaction product into E.coli DH5 alpha using standard transformation protocols and grow the transformed bacteria on an LB plate containing 50 micrograms per milliliter kanamycin.
After verifying the sequences of the short tandem target mimic fragments by terminator cycle sequencing, isolate the PVX VBMS plasmids from the validated clones and transform them into the appropriate Agrobacterium strains. To inoculate four-week-old in vitro potato plants with Agrobacterium containing the PVX VBMS plasmids, pick positive transformants containing PVX VBMS vectors and inoculate the transformants in 50 milliliters of liquid LB containing 50 micrograms per milliliter of kanamycin and 50 micrograms per milliliter of rifampicin for 16 hours at 28 degrees Celsius and 220 revolutions per minute. During the incubation, streak positive Agrobacterium colonies onto at least two new LB plates containing kanamycin and rifampicin and grow the cultures at 28 degrees Celsius for one day.
When the optical density at 600 nanometers reaches 1.0, collect the Agrobacterium liquid culture by centrifugation and resuspend the pellet in a sufficient volume of infiltration buffer to adjust the optical density at 600 nanometers to 1.0. After a six-hour incubation at room temperature, load a one milliliter needleless syringe with the Agrobacterium suspension and use one hand to flip and hold an expanded leaf from one potato plant. Using one finger, support the leaf lamina from the adaxial side at the desired site of infiltration.
And holding the syringe vertical to the leaf surface with the other hand, infiltrate 100 to 200 microliters of Agrobacterium culture into the abaxial side of the lamina. Next, use a toothpick to collect colonies from the Agrobacterium culture plates and to scratch the stem surface of the first one or two internodes of the infiltrated potato plants and the epidermis of the stem, then grow the infiltrated plants in the greenhouse for two to four weeks. When phenotypes appear, use scissors to collect samples with phenotypes from the VBMS and control plants.
Isolate the total RNA from the collected tissues and use reverse transcriptase PCR to analyze the microRNA and target mRNA expression in each sample. In these images, PVX short tandem target mimic 165/166 potato plants with ectopic growth of leaf tissues from the abaxial side of leaf lamina along the veins can be observed. More severe phenotypes such as trumpet-shaped leaf formation have also been observed.
In contrast, no phenotypical abnormalities are observed in PVX control plants, demonstrating that the VBSM system is effective in suppressing endogenous microRNA function in tetraploid potato plants. PVX short tandem target mimic 165/166 potato plants exhibit ectopic leaf to tissue growth from the abaxial side of the leaf lamina along the lamina veins, demonstrating that the PVX VBMS system can be applied to other potato varieties, including major potato cultivars. It is essential to simultaneously infiltrate the leafs and to inoculate the stem of the same plant with the same Agrobacterium culture.
Transgenic approaches to stably introducing target mimic molecules into potato plants are recommended when the silencing state of the targeted microRNAs needs to be maintained in the offspring generations.
