FIG 1.

Functional analyses of CpG and UpA dinucleotide constraints in SD-PPV. (A) Histogram showing the mean ± SD of observed-to-expected (odds) ratios for different dinucleotides in potyvirid genomes. (B) Schematic representation of the SD-PPV chimera (each box represents a viral cistron) and its two parental viruses (PPV-R and PPV-D). Segments (F1 to F5) and restriction sites used to build the different SD-PPV derivatives are indicated. (C) Images taken with an epifluorescence microscope of leaves of N. benthamiana plants inoculated with the indicated mutant viruses (F4_) and their parental control (SD-PPV). Inoculated leaves at 6 days postinoculation (dpi) and upper noninoculated leaves at 10 and 20 dpi are shown. Bars, 5 mm. (D) PPV CP-specific immunoblot analyses of extracts from upper noninoculated N. benthamiana leaves (four individual plants) infected with the indicated mutant viruses (F4_) and their parental control (SD-PPV), at 10 and 20 dpi. (E) PPV CP-specific immunoblot analyses of extracts from upper noninoculated P. persica leaves (four individual plants) infected with the indicated viruses, at 15 dpi. Membranes stained with Ponceau red showing the RubisCO large subunit were included as loading controls. Bar graphs showing the mean ± SD (n = 4) of immunoblot signals in arbitrary units are shown on the right (for comparison, we considered the average for SD-PPV to be 1). Different letters indicate significant differences (P < 0.05), by one-way analysis of variance (ANOVA) and Tukey’s honestly significant difference (HSD) test.