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Background  Virus-induced gene silencing (VIGS) is a useful tool for functional characterizations of plant genes. However, the penetrance of VIGS varies depending on the genes to be silenced, and has to be evaluated by examining the transcript levels of target genes.    Results  In this report, we report the development of a novel VIGS vector that permits a preliminary assessment of the silencing penetrance. This new vector is based on an attenuated variant of  Turnip crinkle virus  (TCV) known as  CPB  that can be readily used in  Arabidopsis thaliana  to interrogate genes of this model plant. A  CPB  derivative, designated  CPB1B , was produced by inserting a 46 nucleotide section of the  Arabidopsis PHYTOENE DESATURASE  ( PDS ) gene into  CPB , in antisense orientation.  CPB1B  induced robust  PDS  silencing, causing easily visible photobleaching in systemically infected  Arabidopsis  leaves. More importantly,  CPB1B  can accommodate additional inserts, derived from other  Arabidopsis  genes, causing the silencing of two or more genes simultaneously. With photobleaching as a visual marker, we adopted the  CPB1B  vector to validate the involvement of  DICER-LIKE 4 (DCL4)  in antiviral defense against TCV. We further revealed the involvement of  ARGONAUTE 2 (AGO2)  in  PDS  silencing and antiviral defense against TCV in  dcl2drb4  double mutant plants. These results demonstrated that  DOUBLE-STRANDED RNA-BINDING PROTEIN 4 (DRB4) , whose protein product (DRB4) commonly partners with DCL4 in the antiviral silencing pathway, was dispensable for  PDS  silencing induced by  CPB1B  derivative in  dcl2drb4  double mutant plants.    Conclusions  The  CPB1B -based vector developed in this work is a valuable tool with visualizable indicator of the silencing penetrance for interrogating  Arabidopsis  genes, especially those involved in the RNA silencing pathways.

Simultaneous silencing of two different Arabidopsis genes with a novel virus-induced gene silencing vector