FIGURE 5.

Detection of the synthesized viral RNA in the strains transfected using protoplasts as the receptor. (A) SsOLV4 RNA was detected by RT-PCR amplification from the transfected subcultures. Total RNA of each strain was used to synthesize cDNA. Gene actin was amplified as an internal control using primers ActinF and ActinR. Primers are listed in Supplementary Table S1. DNA was stained with ethidium bromide. Size of the DNA ladder standard is indicated in base pairs. (B) Northern hybridization to confirm the presence of SsOLV4. Total RNA from the strains was extracted from S. sclerotiorum mycelia grown for 2 days on PDA covered with cellophane. Each RNA sample was about 20 ng in total. Marker RL10000 (Takara) was detected using probe control DNA in alkaline Direct Labeling System (GE Healthcare). Lower panels are ethidium bromide-stained gels showing rRNA. The probe (location shown in Figure 2A) was synthesized with primers OMVFA and OMVR2 using PCR and directly labeled with alkaline phosphatase and detected by CDP-Star (GE).