Preparation of total-RNA or ssRNA samples and cDNA synthesis for RT-PCR.

For nucleic acid extraction, frozen mycelia were pulverized using liquid nitrogen and a mortar and pestle. Total RNAs were extracted with Iso-RNA lysis reagent (5 Prime, Hamburg, Germany). Extracted total RNA was treated with DNase I (TaKaRa Bio, Shiga, Japan) to remove genomic DNA according to the manufacturer's instructions. These total-RNA samples were precipitated with ethanol and resuspended in DEPC-treated water. Next, 5 μg of total RNA of each sample was used to synthesize first-strand cDNA with an oligo(dT)₁₈ primer and Moloney murine leukemia virus (MMLV) reverse transcriptase (Promega, Madison, WI) according to the manufacturer's protocols. All synthesized cDNAs were diluted 1:10 with nuclease-free water for RT-PCR. To isolate the ssRNA fraction, total-RNA extracts were precipitated with LiCl to a final concentration of 2 M. Samples were precipitated after incubation at 4°C for 2 h. ssRNA pellets were washed in 75% ethanol and dissolved in RNase-free water. First-strand cDNA synthesis was conducted with the GoScript reverse transcription system (Promega, Madison, WI) using virus strand-specific primers (FsRT Fw and FsRT Rv for minus and plus strands, respectively) and oligo(dT)₁₈ primer, using 2 μg of ssRNA.