Fig 1: (a) Symptom development in Nicotiana benthamiana plants infected with GFLV strain GHu or GHu-1EK802G, an asymptomatic mutant of GFLV-GHu in which lysine 802 of protein 1EPol was mutated to glycine, and their corresponding version for which protein 1E was tagged with a V5 epitope. Insets show close-ups of vein clearing symptoms (first and second panels) and asymptomatic leaves (third and fourth panels) at six dpi. (b) Detection of protein 1EPol:V5 accumulation in N. benthamiana leaf tissue systemically infected with wild-type (-) or V5-tagged 1EPol (+) GFLV strains GHu and F13-1EC?1, and mutant GHu-1EK802G by western blot with an anti-V5 antibody. TSP from a mock-inoculated N. benthamiana were used as a control. (c) Analysis of affinity purified 1EPol complexes in TSP from N. benthamiana leaves systemically infected with GFLV-GHu or GFLV-GHu-1E:V5 by western blot. TSP were extracted in lysis buffer 4 amended with 1X Halt protease inhibitor cocktail (Thermo Fisher Scientific) and further diluted 1 : 5 in DTT-free lysis buffer. Inputs were affinity purified with V5 polyclonal antibody (Invitrogen PA1-993) conjugated to Protein A Dynabeads (Invitrogen). The top row of lane labels indicates (e) elution products or (i) input fractions of the affinity purification. The second row indicates V5-tagged (+) or non-tagged (-) GFLV-GHu. The third row indicates the concentration of DTT included in the lysis buffer. The fourth row indicates whether the V5 polyclonal antibody used for affinity purification was untreated (-) or cross-absorbed against TSP from healthy N. benthamiana tissue (+). Proteins were probed with a polyclonal anti-V5 antibody (Invitrogen PA1-993, non-cross-absorbed) and a goat anti-rabbit alkaline phosphatase-conjugated secondary antibody (Invitrogen T2191) and developed with 1-Step NBT/BCIP solution. The predicted molecular weight of (i) V5-tagged 1EPol (93 kDa) is indicated with a red arrowhead, and (ii) a putative V5-tagged 1DProEPol (117 kDa) or 1CVPg1DProEPol (120 kDa) precursor is indicated with a green arrowhead. Bottom panels show Ponceau staining of the RuBisCO large subunit. Molecular weight standards (kDa) are shown by black arrowheads.