Potato Virus X-Based MicroRNA Silencing in Potato Plants

Take an in vitro-grown potato plant.

Next, use recombinant Agrobacterium, a plant pathogen containing a transfer DNA (T-DNA).

The T-DNA carries a full-length potato virus X genome, engineered to encode a non-coding RNA to sequester specific plant microRNAs.

Infiltrate the leaves with the bacteria and scratch the stem with a toothpick coated with the bacteria.

At the inoculation site, the bacteria excise the T-DNA and transfer it as a single-stranded DNA into plant cells.

Using the host machinery, the T-DNA produces viral genomic RNA, which replicates and expresses non-coding RNAs and viral proteins.

The non-coding RNA binds to the target plant microRNAs, preventing them from binding their target mRNAs and triggering developmental defects.

Meanwhile, the viral genomic RNAs form ribonucleoprotein complexes that spread systemically through plasmodesmata to enable microRNA silencing throughout the plant.

Leaf abnormalities, including ectopic outgrowths and trumpet-shaped leaves, indicate the developmental role of the silenced microRNAs.

When the optical density at six hundred nanometers reaches one point zero, collect the Agrobacterium liquid culture by centrifugation, and resuspend the pellet in a sufficient volume of infiltration buffer to adjust the optical density at six hundred nanometers to one point zero.

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 one hundred to two hundred 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.