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

Abstract

Source: Shi, J., et al. Screening and Identification of RNA Silencing Suppressors from Secreted Effectors of Plant Pathogens. J. Vis. Exp. (2020)

This video demonstrates a co-infiltration assay for identifying pathogen effector proteins that suppress RNA silencing in Nicotiana benthamiana 16c. A fluorescent reporter and a candidate effector gene are delivered into plant leaves via Agrobacterium. Normally, RNA silencing degrades reporter transcripts, reducing fluorescence. Suppressor effectors block this response, preserving fluorescence. This assay offers a rapid, visual method to screen microbial effectors that interfere with host defense.

Protocol

Co-infiltration Nicotiana benthamiana leaves

1. Mix equal volumes of an Agrobacterium culture containing 35S-GFP with an Agrobacterium culture containing 35S- cucumber mosaic virus suppressor 2b (CMV2b), putative effector, or empty vector (EV).
2. Carefully and slowly infiltrate the mixed agrobacterium suspensions on the abaxial sides of N. benthamiana 16c leaves using a 1 mL needleless syringe.
3. Remove the remaining bacterial suspension from the leaves with soft tissue wipers and circle the margins of the infiltrated patches with a maker pen.
4. Leave the infiltrated plants in the growth chamber under the same growth conditions.
   
   NOTE: For safety and health reasons, protective eye goggles and a mask should be worn during infiltration. To prevent cross-contamination, gloves should be changed or sterilized with ethanol between infiltrations. Normally at least 1 mL of agrobacterium suspensions per leaf are needed for infiltration. For systemic silencing, at least two leaves will be required for infiltration.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
UV lamp	Analytik Jena	UVP B-100AP	Observation