FIG 1.

Mutual exclusion of SYNV variants tagged with GFP and RFP in N. benthamiana. (A) Schematic diagrams showing rSYNV antigenomes engineered to express GFP (rSYNV-GFP) and RFP (rSYNV-RFP). (B) Confocal microscopy z-stack images showing spatial separation of GFP and RFP fluorescent foci in upper systemically infected leaves of N. benthamiana plants taken at 12 days postinoculation (dpi) with leaf extract containing mixtures of rSYNV-GFP and rSYNV-RFP. The bottom panels show higher magnifications of the boxed areas. Bar, 100 μm. (C) Confocal microscopy z-stack images of contiguous GFP and RFP foci in N. benthamiana leaf tissue at 15 dpi with agrobacterial mixtures harboring rSYNV-RFP and rSYNV-GFP derivatives. Right panels show magnified views of the boxed sectors on the left to highlight the exclusion boundaries of rSYNV-GFP and rSYNV-RFP in adjacent cells of two foci. Bar, 50 μm. (D) GFP and RFP fluorescence in upper leaves of N. benthamiana plants after an initial sap inoculation with one SYNV variant or mock inoculation, followed 12 days later with a challenge inoculation with the other SYNV variant indicated on the left of the panels. Images were captured with a stereo fluorescence microscope with GFP or RFP channels at 20 days after primary inoculation and again 20 days after challenge inoculation. 1st, primary inoculum; 2nd, challenge inoculum. Bar, 2 mm. (E) Confocal microscopy of N. benthamiana leaves after initial sap inoculation with rSYNV-RFP, followed by a PVX-GFP challenge inoculation 6 days later. The images show overlapping GFP and RFP fluorescence in upper infected leaves taken at 6 days after PVX infection. Bar, 100 μm.