Correlation between P19 protein accumulation and levels of Nef specific siRNAs. (A) ELISA of leaf extracts from Nicotiana benthamiana plants agroinfiltrated with Nef, Nef/TBSV-P19 and Nef/AMCV-P19 was performed using a rabbit polyclonal antibody against TBSV-P19. Leaves were collected at 9 d.p.i. and plant extracts were normalized for total soluble protein (TSP). Fifty micrograms of TSP were loaded in each ELISA plate well. Values are means of triplicate determinations ± standard errors of the means. C-: leaf extract of plants infiltrated with infiltration buffer. (B) Detection of Nef specific siRNAs. Total RNA (15 μg) extracted from leaves agroinfiltrated with Nef, Nef/AMCV-P19, Nef/TBSV-P19, and from mock infiltrated plant used as a control (WT), was separated on denaturing 15% (w/v) polyacrylamide gel with 8 M Urea, stained with ethidium bromide to display relative amounts of rRNA and transferred to a positively charged nylon membrane. Nef specific digoxigenin-labelled RNA (+) (618 nt) was used as probe. M: si RNA low molecular weight RNA marker (synthetic siRNA duplexes 17, 21 and 25 bp).