Fig 6. VCS knockdown brings down PVA^WT expression level to that of PVA^WD.

(A, B) N. benthamiana leaves were infiltrated with Agrobacterium carrying a silencing vector (pHG-VCS) expressing a hairpin RNA targeting VCS (OD₆₀₀ = 0.4). An empty Hellsgate plasmid (pHG-CTRL) was used as a control. Agrobacterium carrying PVA^ΔHCPro, PVA^WD and PVA^WT constructs were co-infiltrated (OD₆₀₀ = 0.01) with Agrobacterium carrying 35S-FLUC (OD₆₀₀ = 0.01) for normalization of the virus-derived RLUC values. Normalized RLUC (A) and PVA RNA (B) levels were quantitated at 4 dpi. (C) Normalized RLUC and (D) RNA values derived from non-replicating PVA RNAs: PVA^{ΔGDD-ΔHCPro}, PVA ^{ΔGDD-HCProWD} and PVA ^{ΔGDD-HCProWT}. VCS silencing was done similarly as in (A). Agrobacterium carrying PVA^{ΔGDD-ΔHCPro}, PVA ^{ΔGDD-HCProWD} and PVA ^{ΔGDD-HCProWT} constructs were co-infiltrated (OD₆₀₀ = 0.05) with Agrobacterium carrying 35S-FLUC (OD₆₀₀ = 0.01) for normalization of the virus-derived RLUC values. Samples were collected at 3 dpi. The number of plants per experiment was 6 in (A, B) and 3 in (C). Statistical significance was assessed using student’s t-test. Different letters above the bars indicate a statistically significant difference (P < 0.05).