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