A. Transgenic Nicotiana benthamiana plants expressing GFP (1) are infiltrated (syringe) with an Agrobacterium suspension containing a GFP construct that induces GFP-RNA silencing. After 10 to 17 days the GFP signal is silenced and chlorophyll red fluorescence is observed under ultraviolet (UV in purple star) light at 474 nm (2). A second infiltration or inoculation is performed on the plant with an infectious clone or a transcript of a virus expressing a putative viral suppressor (3). If the viral suppressor is able to revert silencing the plant will show GFP fluorescence again under uv light, especially in the upper tissue. This routine method can be used to test for differences in viral strains, monitoring simultaneously virus movement and suppressor activity.
B. Coinfiltration of Agrobacterium expressing GFP with a culture containing a construct encoding the “putative viral suppressor” onto non-transgenic N. benthamiana. The illustration shows leaf images to be expected upon UV illumination of a mock-infiltrated leaf with an Agrobacterium culture without the GFP construct (1), a leaf infiltrated with the GFP expressing construct (2), and a leaf coinfiltrated with constructs expressing GFP and the candidate suppressor (3). At 10 days after infiltration the GFP signal usually decays due to RNA silencing (red fluorescence of chloroplasts only), but if the protein expressed from the coinfiltrated construct has suppressor activity, the GFP signal (green fluorescence) will remain strong as illustrated in 3.

A double-stranded (ds) RNA molecule is targeted by a DICER_LIKE protein (DCL) with assistance from a dsRNA binding protein (DRB), and cleaved (indicated by scissors) into 21-24-nt siRNAs with the signature 2-nt overhang (short staggered duplex arrows). Addition of methyl groups (CH₃) to the ends of the siRNA is performed by HEN1, siRNAs that are not methylated end up polyuridylated and degraded (curved white arrow) by the exosome. The methylated siRNA is recognized by the RISC-AGO holocomplex and one of the siRNA strands is incorporated into the complex (short white arrow). The programmed RISC targets the cognate mRNA (thick blue arrow) and hydrolyzes it (scissors) at a central location on the siRNA sequence. The cleaved products are either used as templates for siRNA amplification (curved blue arrow) or they are degraded by the XRN4 5’-3’ exonuclease; blue lines without arrows indicate RNA remnants. The amplification cycle requires the activity of RDR, SDE3, SGS3, DCL-DRB and AGO (in RISC). This amplified siRNA then presumably serves as a silencing signal. For each step a list of suppressors (brown characters) that interfere with silencing is provided in brackets (see text for details on origin and function); FIERY1 represents an endogenous suppressors. A group of viral (and the host rgs-CAM) suppressors for which other than RNA binding a specific function has not yet been precisely identified, is listed at the very right. Also is indicated that viral siRNAs can be used to program RISC against host RNA.