Fig. 4.

Schematic representation of genome organization and infectivity assay of the parental CymMV-M1 and CymMV-M2 and the derived TGBp1 and TGBp3 chimeric constructs in N. benthamiana. (A–H). Sequences corresponding to pCymMV-M1 and pCymMV-M2 are indicated by gray and white rectangles, respectively. Clones competent in protoplast accumulation and systemic infection are indicated by +, and the ratio of systemic infected to total inoculated plants is indicated. Systemic infection was detected 2 weeks post-inoculation by RT-PCR. (I) Protoplast infectivity was detected 24 h post-inoculation by northern blot hybridization, and the ribosomal RNA used for a loading control are indicated. Genomic RNA (G), TGBp, and CP subgenomic RNA are indicated. The pCymMV-R- used as a negative control is illustrated in Fig. 1. The average percentage of relative real-time RT-PCR quantification (from 3 independent experiments) of CymMV RNA from CymMV clone-infected protoplasts at 24 h post-inoculation is indicated below the gels. The accumulation of pCymMV-M1 was set at 100% for relative quantification. Numbers at the left correspond to positions of marker RNAs (sizes in 1000 nucleotides) analyzed in the same gel. The primers CymMV F3783 and CymMV CPR used in construction all chimeric viruses are indicated. Other primers used in construction individual chimeric viruses are in Supplementary Table S1. Numbers at the left correspond to positions of marker RNAs (sizes in 1000 nucleotides) analyzed in the same gel.