Fig. 3

a Systemic symptoms (30 dpi) induced in leaves of N. benthamiana inoculated with DNA clones of (1, 2) GVA and (4) GVB genetic variants P163-M5 and 953-1, and the wild-type of these variants (3, 5), respectively. (6) Virus-free N. benthamiana. b dsRNA extracted from systemically virus infected symptomatic leaves of N. benthamiana inoculated with (1–3) GVA and (5, 6) GVB genetic variants P163-M5 and 953-1, using (1, 2, 5) DNA clones and (3, 6) the wild-type of these variants, respectively. Lane 4 shows dsRNA extracted from N. benthamiana inoculated with the mild wild-type GVA, variant P163-1 (Goszczynski and Jooste 2003a). Arrows point to dsRNA fragments clearly visible in EtBr-stained 6 % polyacrylamide gels. c Western Blot reaction of GVA- and GVB-specific rabbit antisera, produced to capsid proteins of these viruses (Galiakparov et al. 2003), with extracts from (1–6) virus-infected N. benthamiana inoculated with DNA clones of (1, 2) GVA and (4, 5) GVB genetic variants P163-M5 and 953-1, and (3, 6) the wild-type of these variants, respectively. Lanes 7 show lack of reaction of these antisera with extracts from virus-free plants. (M) Prestained MW marker (38 kDa, Sigma). Virus-specific rabbit antibodies were detected by incubation of nitrocellulose membranes with goat anti-rabbit alkaline phosphatase conjugate (GAR-AP) (Sigma). The mixture of Naphthol AS-MX phospohate (Sigma) and Fast Red TR salt (Sigma) in 0.2 M Tris–HCl buffer, pH 8.2, was used as substrate for the alkaline phosphatase (Goszczynski et al. 1997)