Fig. 1

Co-expression of heterologous VSRs enhances accumulation of GFP expressed from PVX-based vectors in N. benthamiana. (A) Genome organization of the original PVX vector (pPVX: GFP) and its deletion derivatives, namely, pP1:GFP (ΔCP) and pP2:GFP (ΔTGB-ΔCP). Key vector components include the Cauliflower mosaic virus 35 S promoter (35 S), RNA-dependent RNA polymerase (RdRp), triple gene block (TGB), coat protein (CP), nopaline synthase terminator (NOS), T-DNA left and right borders (LB and RB), and a subgenomic promoter (SGP). (B) Schematic representation of pHREAC-based binary vectors used to co-express heterologous VSRs. The TZSV NSs-coding sequence was replaced by the TBSV P19-coding sequence (pH: P19) or the TCV P38-coding sequence (pH: P38), each under the control of the CaMV 35 S promoter and 35 S terminator (35T). (C) N. benthamiana leaves were agroinfiltrated with pPVX: GFP, pP1:GFP, or pP2:GFP and co-infiltrated with a binary vector expressing P19 (TBSV), P38 (TCV), NSs (TZSV), or an empty vector (EV) at a 1:1 ratio. GFP fluorescence was visualized under UV light at 5 dpi. Representative images from three independent experiments with at least three biological replicates (n ≥ 9) are shown. (D) Western blot analysis of total protein extracts (10 µg per sample) from infiltrated leaves using an anti-GFP antibody. Non-treated (NT) samples, serving as mock controls, were included as negative controls. The Rubisco large subunit stained with Ponceau S (PS) was used as a loading control. GFP signal intensity was quantified using ImageJ. Data represent mean ± SD from one representative experiment out of three repeats (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test; different letters indicate significant differences (p < 0.05).