FIGURE 6.

The infectivity of citrus tristeza virus (T36CA‐V1.3) with two amino acid substitutions introduced into the T36CA‐V1.1 encoded p65. Conserved nucleotides from the T36CA population were engineered into the p65 gene of pT36CA‐V1.1 to replace two nonconserved/nonsynonymous nucleotide variants. The resulting construct, pT36CA‐V1.3, encoding a p65 that contained two amino acid substitutions, N118S and S158L, was agroinfiltrated to transgenic (HC‐Pro‐expressing) Nicotiana benthamiana plants. (a) Representative images of green fluorescent protein (GFP) fluorescence observed in the infiltrated and systemic leaves of pT36CA‐V1.3‐inoculated plants at 10 days postinoculation (dpi) and 4 weeks postinoculation (wpi), respectively. A systemic leaf with vein chlorosis at 5 wpi, like those observed in Hyb1‐infected plants (see Figure 3b), is shown to the right of the GFP‐fluorescing systemic leaf. (b) Assessment of virus accumulation in the systemic leaves of transgenic (HC‐Pro) N. benthamiana plants at 5 wpi. The A₄₀₅ value for each virus/treatment indicated on the x axis is the average for eight individual samples in total from two representative experiments (with error bar representing SE). (c) A transmission electron micrograph with scale bar (as indicated), showing a virion‐like particle of T36CA‐V1.3. (d) Examination of bark peels obtained from the new branches of T36CA‐V1.3 virion preparations‐inoculated Citrus  macrophylla plants by wide‐field fluorescence microscopy, revealing the presence of GFP fluorescence that increased in intensity and abundance from 5 to 10 wpi