Fig 1. HCpro, the potyviral suppressor of RNA silencing, is the protein responsible for PVA-induced granules (PGs).

P0^YFP was co-expressed with PVA^WT (A) and alone (B), and P1^YFP (C) and P2^YFP (D) were expressed with PVA^WT in Nicotiana benthamiana leaves using agroinfiltration and imaged by confocal microscopy three days later. RFP was expressed in A-D to visualize the nucleus. In (A- D) the left panel shows the YFP signal and the right panel an overlay view of YFP, nuclear RFP and the bright field (BF) as Z-stack projections. Arrowheads point out P0^YFP-labeled PGs in (A). Scale bar; 10 μm. (E) Frequency of cells/mm² containing PGs in leaves expressing P0^YFP together with PVA proteins P1, HCpro, CI, 6K2, VPg, NIa, NIb or CP. PVA^ΔGDD was expressed as a positive and GUS as a negative control for PG induction. (F) Frequency of cells/mm² containing PGs in leaves expressing P0^YFP together with GUS, PVA^ΔGDD or PVA^ΔHCΔGDD (left panel) or GUS, PVA or PVA^ΔHC (right panel). (G) Frequency of cells/mm² showing PGs in leaves expressing P0^YFP with PVA^WT, PVA^CPmut, PVA^ΔGDD or HCpro plotted as a function of time, (H) a western blot analysis of HCpro accumulation in PVA^WT, PVA^CPmut, PVA^ΔGDD and HCpro samples using anti-HCpro PAb four days post infiltration and (I) virus derived RLUC activities analyzed in the same samples as in (H), again plotted as a function of time (days post infiltration). Modified PVA infectious cDNA constructs used in this study are schematically presented in (S1 Fig). All quantitative data is presented as means and the error bars indicate the standard deviations. (p < 0.01 **, p < 0.05 *).