Co-Infiltration Assay for Screening Pathogen Effectors that Suppress Plant RNA Silencing

Take a mixture containing equal volumes of two Agrobacterium tumefaciens suspensions, each carrying a plasmid-one encoding GFP, and the other encoding an effector protein.

Puncture a hole and use a needleless syringe to infiltrate the mixture into the lower surface of healthy, fully expanded Nicotiana benthamiana 16c leaves.

Blot any excess suspension with soft tissue to prevent bacterial overgrowth.

Mark the infiltrated areas and incubate the plants under optimal growth conditions.

Agrobacterium transfers the plasmid DNAs into the plant cells, where the GFP and effector genes are transcribed into mRNAs and translated into proteins.

The plant’s defense system uses RNA silencing, where duplex siRNAs are loaded into RISC. The passenger strand is removed, and the guide strand directs the complex to degrade target GFP mRNA.

However, the effector proteins bind to siRNAs, preventing their association with RISC and allowing GFP fluorescence to persist.

This enables the identification of pathogen effectors that suppress plant RNA silencing.

Mix equal volumes of an Agrobacterium culture containing 35S green fluorescent protein with an Agrobacterium culture containing 35S cucumber mosaic virus suppressor 2b, putative effector, or empty vector. Using a 1 milliliter needleless syringe, carefully and slowly infiltrate the mixed Agrobacterium suspensions on the abaxial sides of Nicotiana bethamiana 16c leaves.

Remove the remaining bacterial suspension from the leaves with soft tissue wipes, and circle the margins of the infiltrated patches with a marker pen.