Journal: PLoS Biology

Article Title: Viruses Roll the Dice: The Stochastic Behavior of Viral Genome Molecules Accelerates Viral Adaptation at the Cell and Tissue Levels

doi: 10.1371/journal.pbio.1002094

Figure Lengend Snippet: (A) The genome structure of wild-type ToMV and its derivatives used in the experiments. Rectangles indicate open reading frames (ORFs). MT, a methyltransferase-like domain; HEL, a helicase-like domain; POL, a polymerase-like domain of the replication proteins; MP, the cell-to-cell movement protein; and CP, the coat protein. Translation of the genomic RNA produces two replication proteins, 130K and 180K; 130K contains MT and HEL. The 180K is synthesized via the UAG stop codon read-through of 130K to express POL. FP indicates the fluorescent protein genes (YFP or CFP). TLPYFP-CP and TLPCFP-CP carry the YFP and CFP gene, respectively, between the MP and CP coding regions. The expression of the YFP or CFP genes is driven by a duplicated subgenomic promoter (sgp) for the CP. TLPYFP and TLPCFP lack the CP gene. The regions that correspond to the PPYP and P5′M probes, which were used for northern blotting and RNase protection assays, are indicated by bold lines. (B) An example of spatial separation of the two variants during cell-to-cell spread from a coinfected cell in a tobacco leaf. YFP and CFP fluorescence images were obtained separately 44 hours postinoculation (hpi) using a color charge-coupled device (CCD) camera. After converting the color of the CFP fluorescence image to magenta, the image was merged with the YFP fluorescence image without color conversion. The YFP images look green (rather than yellow) because of the transmission range of the emission filter. Coinfected cells were found at the central part of the infected region as white colored cells. (C) The stochastic occurrence of single infections by one of the two co-inoculated variants in protoplasts. Fluorescence images were obtained at 24 hpi and then processed as described above. White arrowheads indicate singly infected cells.

Article Snippet: The cDNA plasmids and cDNA library of the ToMV variants were linearized at the Mlu I restriction site and transcribed using an AmpliCap-Max T7 RNA polymerase kit (CellScript, Madison, Wisconsin, US).

Techniques: Synthesized, Expressing, Northern Blot, Fluorescence, Transmission Assay, Infection