Detection of PVY CP and PVY small interfering RNAs in N. benthamiana agroinfiltrated tissues. (A) Schematic representation of pIRCPPVY used for transient expression by agroinfiltration. A cDNA fragment encoding sense and antisense PVY CP RNA sequences separated by a spacer sequence (Phe) were cloned into binary plant vector pCAMBIA2300. (B) Plants were agroinfiltrated with empty vector or pIRCPPVY and used after 4 days post-infiltration to inoculate PVY. Where no agroinfiltration occurred (right panel), plants were directly inoculated with PVY (PVY) or buffer (healthy). (C) Plants were agroinfiltrated with empty vector or pIRCPPVY and used after 4 days post-infiltration to feed viruliferous aphids (M. persicae). At 14 dpi., sap extracts from upper leaves of single plants were assayed by dot blot using PVY antiserum, and detected using a secondary antibody conjugated to peroxidase. (D) Northern blot analysis of low molecular weight RNAs shows the accumulation of siRNAs in pIRCPPVY-infiltrated leaves. Samples were taken 4 days after infiltration. The blot was hybridised with a 32P-labeled cDNA PVY CP probe. Equivalent loading of samples was shown by staining the gel with ethidium bromide before transfer. The mobilities of oligodeoxynucleotides of the indicated length are shown to the right.