Figure 1.
Disease response in C. annuum ‘Bukang’ inoculated with CMV-Fny, CMV-P1, and CMV reassortants.
A, Disease symptoms of systemic leaves inoculated with the indicated viruses (CMV-Fny, CMV-P1, P1F2F3, F1P2F3, F1F2P3, F1P2P3, P1F2P3, and P1P2F3) or mock inoculated. Pepper cotyledons were inoculated using sap from infected N. benthamiana. Photographs were taken at 21 days post-inoculation. B, Detection of CMV accumulation via enzyme-linked immunosorbent assay (ELISA). Two leaf discs from inoculated cotyledons and the upper leaves were sampled at 28 days post-inoculation. Three independent experiments were each performed in triplicate. a Average of absorbance value and standard errors.
Table 1.
Amino acid differences in protein 1a of CMV-Fny and CMV-P1.
Figure 2.
Schematic diagram of CMV RNA1 chimeric viruses.
White boxes and gray boxes indicate Fny-derived regions and P1-derived regions, respectively. Dotted lines show the exchange position or the common restriction site between CMV-P1 and CMV-Fny used in the construction of the chimeras. R, resistant; S, susceptible.
Figure 3.
Symptoms in C. annuum ‘Bukang’ inoculated with CMV-Fny, CMV-P1, and CMV RNA1 chimeras. A
, Symptoms in C. annuum ‘Bukang’ inoculated with CMV-Fny, CMV-P1, and CMV RNA1 chimeras. Cotyledons were inoculated using sap from infected N. benthamiana. The photographs were taken at 21 days post-inoculation. B, Accumulation of CMV coat protein in inoculated leaves and systemic leaves of peppers was detected using ELISA. Two leaf discs of the inoculated cotyledons and the non-inoculated leaves from each plant were sampled at 21 days post-inoculation. Three independent experiments were each performed in triplicate. aAverage of absorbance value and standard errors.
Figure 4.
Schematic diagram of CMV-Fny- and CMV-P1-derived amino acid substitution mutants.
The positions of the amino acid substitutions are located at the top of each amino acid sequence. Fny- and P1-derived amino acids are indicated in blue and red, respectively. At 21 days post-inoculation, accumulation of CMV was detected by ELISA. +, presence of virus; −, absence of virus; R, resistant; S, susceptible.
Table 2.
Summary of experiments with helicase domain mutant viruses.
Figure 5.
Virus movement and localization studies with CMV clones.
A and B, Localization of CMV-Fny-GFP (A–a, b, c; B–a, b, c), CMV-P1-GFP (A–d, e, f; B–d, e, f), F (iii, vi)-GFP (A–g, h, i; B–g, h, i), and F (-iii, -vi)-GFP (A–j, k, l; B–j, k, l) in C. annuum ‘Bukang.’ Images a to c, d to f, g to i, and j to l in panels A and B indicate optical sections moving down into the leaf from the epidermal cells at the leaf surface to the underlying mesophyll cells. GFP fluorescence was visualized by confocal laser scanning microscopy at 4 (A) and 7 (B) days post-inoculation (dpi). Green indicates GFP fluorescence and red indicates chlorophyll autofluorescence. Scale bars = 50 µm. C, In situ hybridization localization of the CMV-Fny, P1 and F (-iii, -iv) in inoculated leaves of C. annuum ‘Bukang’ at 4 dpi (C–a to e) and 8 dpi (C–f to j). These images are transverse sections of mock (a and f)-, CMV-Fny (b, c, g and h)-, CMV-P1 (d and i)- and F (-iii, -iv) (e and j)-inoculated leaf tissues. The sections of CMV-Fny inoculated leaves shows two scale images, 10X (b and g) and 5X (c and h). Purple color and arrows indicate the infected region. Scale bars = 100 µm.
Table 3.
Quantification of the genomic RNA copies CMV-Fny, CMV-P1 and chimeric viruses in inoculated leaves of C. annuum ‘Bukang’.