A geminivirus-related DNA mycovirus that confers hypovirulence to a plant pathogenic fungus

Abstract

Mycoviruses are viruses that infect fungi and have the potential to control fungal diseases of crops when associated with hypovirulence. Typically, mycoviruses have double-stranded (ds) or single-stranded (ss) RNA genomes. No mycoviruses with DNA genomes have previously been reported. Here, we describe a hypovirulence-associated circular ssDNA mycovirus from the plant pathogenic fungus Sclerotinia sclerotiorum. The genome of this ssDNA virus, named Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), is 2166 nt, coding for a replication initiation protein (Rep) and a coat protein (CP). Although phylogenetic analysis of Rep showed that SsHADV-1 is related to geminiviruses, it is notably distinct from geminiviruses both in genome organization and particle morphology. Polyethylene glycol-mediated transfection of fungal protoplasts was successful with either purified SsHADV-1 particles or viral DNA isolated directly from infected mycelium. The discovery of an ssDNA mycovirus enhances the potential of exploring fungal viruses as valuable tools for molecular manipulation of fungi and for plant disease control and expands our knowledge of global virus ecology and evolution.

Fig. 1.

Hypovirulence-associated traits of strain DT-8 of S. sclerotiorum. (A) Abnormal colony morphology of strain DT-8 grown on a PDA plate at 20 °C for 15 days; (B) hypovirulent phenotype of strain DT-8 as exhibited by infected Arabidopsis thaliana plants, which were maintained at 20 °C for 4 days postinoculation; (C) small sclerotia produced by strain DT-8 on a PDA plate at 20 °C for 30 days; and (D) growth rate of strain DT-8 relative to other strains, bars represent standard deviation from the mean (n = 8). The small letters on top of the bars in D indicate whether the differences are statistically significant (P < 0.05). The RNA virus-infected hypovirulent strain Ep-1PN and its sexual progeny Ep-1PNA367 (virus-free) were used as controls; DT-8VF is a virus-free culture derived by hyphal tipping of strain DT-8, and showed a normal phenotype of S. sclerotiorum.

Fig. 2.

Genomic characteristics and particle morphology of Sclerotinia sclerotiorum hypovirulence associated DNA virus 1 (SsHADV-1). (A) Total DNA extracted from mycelia of strains DT-8 and DT-8VF. DNA samples were fractionated on 1.0% agarose gel. The positions of host genomic DNA, viral DNA, or the large DNA element (LDE) and defective viral DNA or small DNA element (SDE) of strain DT-8 are indicated. Lane M1, λ-Hind III-digested DNA Marker; lane M2, DL2000 DNA ladder marker (TaKaRa). (B) Southern blot analysis of total nucleic acid extracted from mycelia of strains DT-8 and DT-8VF. The forms of viral DNA are indicated as OC dsDNA [open circular double-stranded (ds) DNA], SC dsDNA (supercoiled dsDNA), and circular single-stranded (ss) DNA. A 379-bp DNA fragment of Rep was PCR amplified and labeled with α-³²P dCTP and used as a probe. (C) Viral particles observed under transmission electron microscopy. Particles were purified from mycelia of strain DT-8 and negatively stained with 1% uranyl acetate. (Scale bars, 50 nm.) (D) SDS/PAGE analysis of SsHADV-1 coat protein. Samples collected from fractions corresponding to 25% sucrose of sucrose gradients were subjected to SDS/PAGE. The size of the Coomassie blue-stained protein was estimated by comparison with protein markers. (E) Agarose gel electrophoresis on 1% agarose of DNA extracted from sucrose gradient fractions containing virus particles. (F) Genome organization of SsHADV-1. Functional ORFs (coding for CP and Rep) were displayed as thick arrows. The positions of the potential stem–loop structure, large intergenic region (LIR) and small short intergenic region (SIR) were marked; the stem-loop structure was shown on the right. LIR is also shown in an expanded form to indicate the elements of the bidirectional promoter.

Fig. 3.

Phylogenetic analysis of SsHADV-1. (A) Amino acid sequence alignment of Rep of SsHADV-1 and selected viruses from the genus Mastrevirus in the family Geminiviridae. The conserved motifs (I to VII) were shaded with light gray color; Asterisks indicate identical amino acid residues, and colons indicate similar residues. Conserved motif Ito VII are IRD, RCR-I, RCR-II, RCR-III, RBR, NTP Binding-A, and NTP binding-B, respectively. The Alignment was generated by using CLUSTALX (2.0). Numbers in brackets are the positions of amino acid residues that are not listed. (B) Phylograms of the Rep and CP of SsHADV-1 and selected circular ssDNA viruses in the families Geminiviridae, Nanoviridae and Circoviridae. Multiple alignments of amino acids and tentative phylogenetic trees generation were referred to the method described by Liu et al. (20). The position of SsHADV-1 is indicated by a red star. See Table S1 for abbreviations of virus names and viral protein accession numbers used for phylogenetic analysis.

Fig. 4.

PEG-mediated protoplast transfection with purified SsHDV-1 particles and viral DNA extracted directly from infected mycelium. Colony morphology of the virus-free strain Ep-1PNA367 and its newly transfected isolates. Ep-1PNA367-NT3 and Ep-1PNA367-NT7 were isolated from transfectants with purified viral DNA, whereas Ep-1PNA367-PT2 and Ep-1PNA367-PT6 were isolated from transfectants with viral particles. Colonies were grown on PDA plate at 20 °C for 7 days. (B) Viral DNA extracted from transfected cultures of strain Ep-1PNA367. Lane 1, strain Ep-1PNA367; lane 2, Ep-1PNA367-NT3; lane 3, p-1PNA367-NT7; lane 4, Ep-1PNA367-PT2; lane 5, Ep-1PNA367-PT6; lane 6, strain DT-8; and lane M, λ-Hind III-digested DNA Marker. (C) Viral DNA of newly transfected cultures were confirmed by PCR amplification with primer pair designed from the Rep sequence of SsHADV-1. Lane M, DL2000 DNA ladder marker; lane 1 to lane 6 are as the same as in B.