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Supporting Information Supporting Information Yu et al. 10.1073/pnas.0913535107 Fig. S1. Virulence of S. sclerotiorum strains DT-8 and DT-8VF on detached leaves of rapeseed. The fungal strains were cultured on PDA plates for one day after activation, and mycelial discs, cut from the colony margin, were used as inocula by placing them on the detached leaves. The inoculated leaves were then kept in an incubator (100% RH and 20 °C). Fig. S2. Transmission of hypovirulence-associated traits via dual-culture from strain DT-8 to the virus-free strain DT-8VF. (A) Strain DT-8 was inoculated onto a PDA plate 2 days before inoculation with strain DT-8VF. Following contact with strain DT-8, the colony of strain DT-8VF was converted to a phenotype similar to that of strain DT-8, and slowly extended onto the plate. The colony of strain DT-8VF that grew alone completely covered the plate. Strain DT-8VF was cultured at 20 °C for 5 days. (B) Newly infected isolates of strain DT-8VF lost virulence on Arabidopsis. (C) Viral DNA was extracted from newly infected DT-8VF. Lane M, DL2000 DNA ladder marker Yu et al. www.pnas.org/cgi/content/short/0913535107 1of6 Fig. S3. Characterization of two additional DNA fragments extracted from mycelia of strain DT-8 and viral DNA isolated from viral particles. (A) DNA samples were resuspended in RNase A-containing TE buffer and treated with different nucleases. The DNA samples were hydrolyzed by S1 nuclease and DNaseI but not by either Exonuclease III or Exonuclease I. Lane M, DL2000 DNA ladder marker. The nuclease treatment results suggest that the two additional DNA fragments were either circular ssDNA or linear ssDNA with modified 5′and 3′ ends. The host genomic DNA served as a dsDNA control. (B) DNA samples were resuspended in RNase A-containing TE buffer and treated with ssDNA nucleases S1 or Exonuclease I. The ssDNA form of the genome of M13 phage was used as a control. Lane M, DL2000 DNA ladder marker. Fig. S4. Identification of the viral ORFs (ORFs) and gene expression in the hypovirulent strain DT-8. (A) Northern hybridization analysis of the mRNAs cor- responding to the two ORFs of SsHADV-1. For RNA extraction, strain DT-8 was cultured on PDA plates at 20 °C and mycelia were harvested on the seventh day and the ninth day after inoculation. The RNA samples were extracted using a TRIzol RNA extraction kit (Invitrogen) and fractionated on 1% agrose gel and transferred onto Nylon membrane (Hybond-N+, Amersham Pharmaciabiotech). The PCR amplification products from either CP or Rep were labeled with α-32P dCTP and used as probes. (B) Expression of the CP and Rep mRNAs in the hypovirulent strain DT8 was confirmed by RT-PCR using SsHADV-1 gene-specific primers. RNA from virus-free strain DT-8VF was included as a negative control. Days postinoculation onto PDA plates are indicated at the top of the figure. Actin levels were used as internal control for cDNA amounts. Yu et al. www.pnas.org/cgi/content/short/0913535107 2of6 Fig. S5. Nucleotide sequence alignment of five defective viral DNA sequences with the genomic DNA of SsHADV-1. The large intergenic region (LIR) is highlighted with red color and the small intergenic region (SIR) is highlighted with green color. The complete nucleotide and deduced amino acid sequences of SsHADV-1 are deposited in GenBank under the accession no. NC_013116. The shortened presentations shown in this figure are derived from the coding regions for Rep and CP. Fig. S6. Amino acid sequence alignment of SsHADV-1 CP and a marine putative protein (Marine_PP), a putative protein encoded by a whole genome shotgun sequence (Genbank accession no.: AACY024124290) from a metagenomic data that analyzed the microbial communities from Sargasso Sea (17). The viral CP shares 37.8% identity and 52.6% similarity with this putative protein. Asterisks indicate identical amino acid residues and colons indicate similar residues. Yu et al. www.pnas.org/cgi/content/short/0913535107 3of6 Fig. S7. Transmission of SsHADV-1 from Sclerotinia sclerotiorum strain DT-8 to the vegetatively incompatible strain Ep-1PNA367R.(i) Vegetative incompatible interaction between strain Ep-1PNA367R (a) and strain DT-8VF (b). Colonies were grown on a PDA plate for one week; red arrow indicates the interaction zone. Photograph was taken from the reverse side of plate. (ii) Dual culture of strain DT-8 (c) and strain Ep-1PNA367R (a) on a PDA plate. Colonies of strain DT-8 and strain Ep-1PNA367R were grown on plates for 9 days and 7 days, respectively; red arrow indicates the interaction zone indicative of vegetative incompatibility. (iii) Colonies of strain DT-8 (c), strain DT-8VF (b), strain Ep-1PNA367R (a) and newly infected isolate of strain Ep-1PNA367R (d) developed in hygromycin amended PDA plate (50 μg/mL). Strain DT-8 and newly infected isolate of strain Ep-1PNA367R were developed in plate for 4 days, whereas strain Ep-1PNA367R and strain DT-8VF were developed in plate for 1 day. Both Ep-1PNA367R and its newly infected isolate could grow on hygromycin amended PDA plate, but strain DT-8 and strain DT-8VF could not. (iv) Transmission of hypovirulence from newly infected isolate of strain Ep-1PNA367R (d) to strain Ep-1PNA367R (a). Colonies of newly infected isolate of strain Ep-1PNA367R and strain Ep-1PNA367R were grown on PDA plate for 10 days and 7 days, respectively. (v) Colonies of newly infected isolate of strain Ep-1PNA367R (d) and strain Ep-1PNA367R (a), colonies were grown on PDA plates for 7 days. (vi) Debilitation of virulence of newly infected strain Ep-1PNA367R on Arabidopsis. (vii) Viral DNA was extracted in newly infected strain Ep-1PNA367R. Lane M, λDNA digested by Hind III; lane 1, newly infected strain Ep-1PNA367; lane 2, Ep-1PNA367R; and lane 3, hypovirulent strain DT-8. Yu et al. www.pnas.org/cgi/content/short/0913535107 4of6 Table S1. Viruses selected for phylogenetic analysis GenBank accession no. Family and Genus Virus Abbreviation Reference Rep CP Geminiviridae Mastrevirus Tobacco yellow dwarf virus TbYDV NP_620726 NP_620725 1 Chickpea chlorotic dwarf virus CpCDV YP_002014712 YP_002014711 2 Bean yellow dwarf virus BeYDV NP_612221 NP_612220 3 Maize streak virus MSV AAK73474 AAK73472 4 Wheat dwarf virus WDV CAC84660 CAC84659 5 Panicum streak virus PanSV AAA62265 AAA62264 6 Begomovirus Ageratum yellow vein China virus AYVV CAD92253 CAD92250 7 Tomato yellow leaf curl Indonesia virus TYLCV YP_699993 YP_699990 8 Papaya leaf curl China virus PaLCuV CAD90114 CAD90111 9 East African cassava mosaic Kenya virus EACMV CAJ78296 CAJ78293 10 Curtovirus Beet curly top virus BCTV AAK59260 AAK59258 11 Beet mild curly top virus BMCTV NP_840040 NP_840037 − Topocuvirus Tomato pseudocurly top virus TPCTV NP_620735 NP_620732 12 Nanoviridae Babuvirus Banana bunchy top virus BBTV ACD62524 ABS12247 13 Abaca bunchy top virus ABTV YP_001661660 YP_001661657 14 Nanovirus Faba bean necrotic yellows virus FBNYV NP_619574 NP_619570 15 Milk vetch dwarf virus MVDV NP_619759 NP_61976 16 Subterranean clover stunt virus SCSV NP_620700 NP_620699 17 Circoviridae Circovirus Canary circovirus CaCV CAC44748 CAD23544 18 Cygnus olor circovirus CyOCV ABU48445 ABU48446 19 Goose circovirus GoCV AAN37996 AAN37998 20 Porcine circovirus 1 PCV1 AAN77859 AAN77864 21 Porcine circovirus 2 PCV2 ABW76686 ABW76687 22 Microviridae Microvirus Enterobacteria phage alpha3 phage NP_039590 NP_039597 23 1. Morris BA, Richardson KA, Haley A, Zhan X, Thomas JE (1992) The nucleotide sequence of the infectious cloned DNA component of tobacco yellow dwarf virus reveals features of geminiviruses infecting monocotyledonous plants. Virology 187:633–642. 2. Nahid N, et al. (2008) Two dicot-infecting mastreviruses (family Geminiviridae) occur in Pakistan. Arch Virol 153:1441–1451. 3. Liu H, et al. (2009) A novel mycovirus that is related to the human pathogen hepatitis E virus and rubi-like viruses. J Virol 83:1981–1991. 4. Martin DP, et al. (2001) Sequence diversity and virulence in Zea mays of Maize streak virus. Virology 288:247–255. 5. Kvarnheden A, Lindblad M, Lindsten K, Valkonen JP (2002) Genetic diversity of Wheat dwarf virus. Arch Virol 147:205–216. 6. Rybicki EP (1994) A phylogenetic and evolutionary justification for three genera of Geminiviridae. Arch Virol 139:49–77. 7. Xiong Q, Fan S, Wu J, Zhou X (2007) Ageratum yellow vein China virus Is a Distinct Begomovirus Species Associated with a DNAbeta Molecule. Phytopathology 97:405–411. 8. Tsai WS, Shih SL, Green SK, Akkermans D, Jan FJ (2006) Molecular characterization of a distinct tomato-infecting begomovirus associated with yellow leaf curl diseased tomato in Lembang, Java island of Indonesia. Plant Dis 90:831. 9. Wang X, Xie Y, Zhou X (2004) Molecular characterization of two distinct begomoviruses from Papaya in China. Virus Genes 29:303–309. 10. Bull SE, et al. (2006) Genetic diversity and phylogeography of cassava mosaic viruses in Kenya. J Gen Virol 87:3053–3065. 11. Hormuzdi SG, Bisaro DM (1993) Genetic analysis of beet curly top virus: evidence for three virion sense genes involved in movement and regulation of single- and double-stranded DNA levels. Virology 193:900–909. 12. Briddon RW, Bedford ID, Tsai JH, Markham PG (1996) Analysis of the nucleotide sequence of the treehopper-transmitted geminivirus, tomato pseudo-curly top virus, suggests a recombinant origin. Virology 219:387–394. 13. Vishnoi R, Raj SK, Prasad V (2009) Molecular characterization of an Indian isolate of Banana bunchy top virus based on six genomic DNA components. Virus Genes 38:334–344. 14. Sharman M, Thomas JE, Skabo S, Holton TA (2008) Abaca bunchy top virus, a new member of the genus Babuvirus. Arch Virol 153:135–147. 15. Timchenko T, et al.
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