FIG. 2.

Agarose gel electrophoretic analysis of viral dsRNAs recovered from transfected C. parasitica colonies 3 weeks posttransfection. Transfected fungal colonies were transferred from regeneration media to PDA plates 7 days posttransfection, cultured for 1 week, transferred to liquid growth media for an additional week of culturing, harvested, and processed to recover dsRNA according to the protocol of Hillman et al. (19). Partially purified viral dsRNA preparations were treated with S1 nuclease (5 U) to digest single-stranded RNA (3) and examined by agarose (0.7%) gel electrophoresis. Full-length viral dsRNAs are the slowest-migrating bands in each lane. The faster-migrating species observed in lanes 7, 15, 17, and 21 correspond to internally deleted defective viral dsRNAs previously identified in hypovirus-infected isolates (2, 35). Lanes marked with an M contain 200 ng of 1-kb DNA ladder (Life Technologies) as relative size markers, with arrows indicating the positions of the 5.1-, 2.0-, and 1.0-kb bands. Lane 1, p29Δ25-243EGFP; lane 2, p29Δ25-243HYG; lane 3, p29Δ25-243EGFP2A; lane 4, p29Δ25-109EGFP; lane 5, p29Δ25-109HYG; lane 6, p29Δ25-109EGFP2A; lane 7, NtNcoI; lane 8, NtNcoIEGFP; lane 9, NtNcoIEGFPp29Δ25-243; lane 10, NtNcoIEGFP2A; lane 11, NtNcoI2AΔp29; lane 12, NtNcoIEGFP2AΔp29; lane 13, Ct2A; lane 14, Ct2APH; lane 15, Ct2APHp29Δ25-243; lane 16, Ctp40[2A]; lane 17, Ctp40[2AEGFP]; lane 18, Ctp40[2AHYG]; lane 19, Δp69bEGFP; lane 20, Δp69bEGFP2A; lane 21, wild-type virus cDNA pLDST.