FIGURE 2.

Viability of MRFV HEL/POL junction as insertion site. (a) Location of primers (sm151 and sm152) used for RT‐PCR detection of MRFV‐PDS₁₂₀ and MRFV‐WT. The primers straddle the HEL/POL junction and amplify 1,164 bp in MRFV‐PDS₁₂₀ and 963bp in wild‐type MRFV. (b) Virus symptoms and the chlorophyll photobleaching phenotype induced by MRFV‐PDS₁₂₀ at 30 dpi, compared to leaves of plants inoculated with MRFV without insert and noninoculated leaves (HC). (c) RT‐PCR analysis of virus accumulation in systemic leaves at 30 days post‐inoculation. The three gels show RT‐PCR detection of MRFV‐PDS₁₂₀ and MRFV‐WT in inoculated plants in three replicated experiments, with RNA from noninoculated plants (HC) and water (H₂O) included as negative control templates; and the MRFV‐PDS₁₂₀ plasmid (PL) serving as a positive control. Blank lanes in MRFV‐PDS₁₂₀ inoculations represent nonsymptomatic plants. M: 100 bp DNA marker. (d) RT‐PCR analysis of insert retention in MRFV‐PDS₁₂₀ at 60 days post‐inoculation. RT‐PCR was repeated at 60 dpi only for RT‐PCR positive (symptomatic/successfully inoculated) MRFV‐PDS₁₂₀ plants in (c) above, with controls and DNA marker included as described above. This was done for all the three replicated experiments. (e) RT‐PCR assays for virus accumulation in tassels and silks for a subset of MRFV‐PDS₁₂₀ symptomatic plants. Controls and DNA marker were included as described above. (f) Transmission electron micrographs of MRFV‐PDS₁₂₀ (left panel) and MRFV‐WT particles (right panel) in maize (Silver Queen) extract