File S1 - A Comparison of Transcriptional Patterns and Mycological Phenotypes following Infection of Fusarium graminearum by Four Mycoviruses

Combined file of supporting figures. Figure S1. Screening of FgVs 1-4-infected PH-1 strains. Virus-infected strains (I: FgV1, II: FgV2, III: FgV3, and IV: FgV4) obtained by fusion experiment were screened by enzyme treatment (A), RT-PCR analysis (B), Southern blot hybridization (C), and AFLP (amplified fragment length polymorphism) fingerprinting (D). Lane M, λ DNA; lane M1, λ DNA-HindIII digested DNA marker; lane M2, 1-kb ladder (Bioneer, Daejeon, Korea). (A) S1 nuclease and DNase I treatment. DK21, 98-8-60, and DK3, donor strains used in this study; lanes 1–8, protoplast fusants. (B) RT-PCR analysis of virus-infected strains. Presence of viral dsRNA was confirmed by RT-PCR amplification with a primer pair designed from the RdRp coding region of each virus. PCR products were separated on 1% agarose gel. No template, negative control; Lanes DK21, 98-8-60, and DK3, positive control; lanes 1–8, protoplast fusants. (C) Southern blot hybridization. Lane 1, PH-1 (wild-type); lane 2, hygromycin B-resistant PH-1 (positive control); lanes DK21, 98-8-60, and DK3, donor strains. Genomic DNAs extracted from protoplast fusants were digested with BamHI and hybridized with PCR fragments from the hygromycin resistance B cassette of pCB1004. (D) AFLP fingerprinting. Lane 1, PH-1 (wild-type); lane 2, hygromycin B-resistant PH-1; lane 3, virus-free donor strain; lane 4, donor strain; lanes 5–11, protoplast fusants. Genomic DNAs of λ DNA and fungal strains were amplified with the primer combinations EcoRI+0/MseI+0 and EcoRI+CA/MseI+GC, respectively. (+0 indicates no selective nucleotides, +CA and +GC indicate selective nucleotides). The molecular weights of the fingerprints ranged from 60–440 nucleotides. Figure S2. Alignment of histone H3 sequences from the Fusarium strains. The fixed nucleotide characters are shaded in green (F. asiaticum; lineage 6) or yellow (F. graminearum; lineage 7). The presence of nucleotides G (position 278) and T (position 279) is differentially fixed for F. asiaticum and F. graminearum, respectively. The GenBank accession numbers of the nucleotide sequences that were used are as follows: NRRL 5883 (AY452815.1), NRRL 6394 (AY452817.1), NRRL 13383 (AY452819.1), NRRL 28063 (AY452816.1), NRRL 28336 (AY452818.1), NRRL 29169 (AY452836.1), and NRRL 31084 (PH-1; AY452852.1). In a previous report [2], we described the molecular identification of DK21 (F. boothii; lineage 3) and DK3 (F. graminearum; lineage 7). Figure S3. Morphology of asci rosettes of F. graminearum PH-1 strains. Each strain was grown on carrot agar for 7 days. After treatment with a Tween-60 solution, all cultures were incubated under UV light for 7 days. Scale bar = 200 µm. Figure S4. Conidial morphology of F. graminearum PH-1 strains. Conidia harvested from 5-day-old CMC cultures were examined with a light microscope. Scale bar = 50 µm. Figure S5. Enriched GO terms according to biological process in the group of DEGs that were down-regulated in response to FgV4 infection. A GO diagram of significantly over-represented GO terms (in the group of DEGs that were down-regulated by FgV4 infection) related to biological process. Figure S6. Enriched GO terms according to cellular component in the group of DEGs that were down-regulated in response to FgV4 infection. A GO diagram of significantly over-represented GO terms (in the group of DEGs that were down-regulated by FgV4 infection) related to cellular component. Figure S7. Comparison between RNA-Seq and microarray data. The numbers of DEGs were compared among three data sets obtained from FgV1 infection. RSeq 120 h indicates RNA-Seq data sampled at 120 hours post infection while Micro 36 h and Micro 120 h indicate microarray data which were obtained at 36 and 120 hours post infection, respectively. (PDF)