Virus induced RNA silencing (VIGS) (a) Construction of PVX/NbRBR1-GFP and PVX/NbPCNA-GFP in PVX/GFP. The PVX genome organisation is shown to encode the RNA-dependent RNA polymerase (RDRP), the triple-gene block (25 K, 12 K and 8 K) and the capsid protein (CP) as well as an insertion of gene of interest (GOI). (b-e) VIGS phenotypes of transgenic Nicotiana benthamiana line pOri2 plants. Plants (25 days old) were mock-inoculated (b) or inoculated with PVX/GFP (c), PVX/NbRBR1-GFP (d) or PVX/NbPCNA-GFP (e). PVX/GFP infected plant showed mosaic and chlorosis at 10 days post-inoculation (dpi) whilst mock inoculated plant remained healthy. Plants challenged with either PVX/NbRBR1-GFP or PVX/NbPCNA-GFP showed local and systemic infection at early stage (7-14 dpi) of viral invasion, but later on (approximately 21 dpi onwards) newly growing leaves showed no viral symptoms. VIGS of NbRBR1 or NbPCNA caused abnormal leaf development and growth retardation. Plants (50 days old) were photographed at 25 dpi. The boxed portions of plants are enlarged to show VIGS-related phenotypes. Leaves of plants with mock inoculation (b) or infected with PVX/GFP (c) at the same developmental stage as these boxed leaves are indicated with an arrow. There leaves were infiltrated with agrobacterium and subsequently used for sqRT-PCR and PCR (see Figures 2 and 3).