Molecular characterization of a novel fusarivirus infecting the plant-pathogenic fungus Alternaria solani

A novel mycovirus belonging to the proposed family “Fusariviridae” was discovered in Alternaria solani by sequencing a cDNA corresponding to double-stranded RNA extracted from this phytopathogenic fungus. The virus was tentatively named “Alternaria solani fusarivirus 1” (AsFV1). AsFV1 has a single-stranded positive-sense (+ssRNA) genome of 6845 nucleotides containing three open reading frames (ORFs) and a poly(A) tail. The largest ORF, ORF1, encodes a large polypeptide of 1,556 amino acids (aa) with conserved RNA-dependent RNA polymerase and helicase domains. The ORF2 and ORF3 have overlapping regions, encoding a putative protein of 522 amino acids (aa) and a putative protein of 105 amino acids (aa), respectively, both of unknown function. A multiple sequence alignment and phylogenetic analysis revealed that AsFV1 could be a new member of the “Fusariviridae”. This is the first report of the full-length nucleotide sequence of a fusarivirus that infects Alternaria solani.

Mycoviruses, also called fungal viruses, are widespread in many simple eukaryotic organisms, such as yeasts, mushrooms, oomycetes, and filamentous fungi, including phytopathogenic fungi [1,9,10]. The majority of fungal viruses typically cause latent infections, but several mycoviruses can cause hypovirulence in their hosts, making them potential biocontrol agents for combating plant fungal disease [11][12][13]. Most fusariviruses infect their hosts without causing any phenotypic changes. However, several fusariviruses, such as a 7.5-kbp mycovirus isolated from Fusarium graminearum strain DK21, are associated with reduced mycelium growth, increased pigmentation, reduced virulence to wheat, and reduced (60-fold) production of trichothecene mycotoxins [14].

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The genomes of viruses in this family are +ssRNA, with sizes of 6-10 kb and one to three ORFs. [15,16].
Here, we report the molecular characterization of a novel +ssRNA mycovirus tentatively named "Alternaria solani fusarivirus 1" (AsFV1). Sequence analysis showed that AsFV1 is closely related to other putative fusariviruses. We think that AsFV1 could be included as a new member of the proposed family "Fusariviridae".

Provenance of the virus material
Alternaria solani strain ZY-D was isolated from a potato leaf infected with early blight in Inner Mongolia Autonomous Region, China, in 2020. Strain ZY-D was cultured on potato dextrose agar (PDA) at 28 °C for 7 days, and the mycelium plugs were placed in potato dextrose broth for culture and shaken at 28 °C and 180 rpm for 5-7 days.
Subsequently, dsRNA was extracted from mycelium using the CF-11 cellulose chromatography method as described by Morris and Dodds, with appropriate adjustments [17]. The sample was then treated with DNase I and S1 nuclease (Takara, Dalian, China) at 37 °C for 40 min to digest DNA and ssRNA, and electrophoresis was performed on a 1.5% (w/v) agarose gel, which was then stained with Goldview and visualized under an UV transilluminator. dsRNA banding in the size range of 5 kbp to 7 kbp was extracted from the gel and purified. Random hexanucleotide primers (dN6 primers: 5′-CGA TCG ATC ATG ATG CAA TGCNNNNNN-3′) and reverse transcriptase were used to construct a cDNA library, which was then sequenced. Specific primers based on the cDNA sequence were used for RT-PCR amplification to fill sequence gaps, and the end sequences were determined using a ligasemediated terminal amplification method [18]. All of the amplified cDNA products were cloned into the vector pMD18-T (Takara, Dalian, China) and introduced into E. coli DH5α for sequencing. Each product was sequenced from at least three independent clones to ensure the accuracy of the sequence. The complete genome sequence of AsFV1 was deposited in the GenBank database with the accession number MW544173.
The National Center for Biotechnology Information (NCBI) database (http:// www. ncbi. nlm. nih. gov/ genom es) was used to perform sequence comparisons and identify ORFs and conserved domains. Multiple sequence alignments were made using the ClustalX program [19] and annotated using the GeneDoc program [20]. A phylogenetic tree was constructed using the NJ method in MEGA 7.0 with 1000 bootstrap replicates [21].
The second and third ORFs have overlapping regions, encoding a putative protein of 522 amino acids (aa) and a putative protein of 105 amino acids (aa) respectively, both of unknown function. The approximate molecular mass of ORF2 and ORF3 are 58.28 kDa and 12.43 kDa, respectively. Amino acid sequence alignment results showed that the virus with the most similarity in ORF2 was Pleospora typhicola fusarivirus 1, with only 48.20% sequence identity, and the virus with the most similarity in ORF3 was ORF-C of Erysiphe necator associated fusarivirus 1, with 32.14% identity. In order to determine the relationship between AsFV1 and other fungal viruses, a phylogenetic tree was constructed based on amino acid sequence of the RdRp domain of AsFV1 and other selected viruses, Fig. 1 (A) dsRNA purified from Alternaria solani was isolated and separated on a 1% agarose gel. Lane M, DNA marker (Trans 15K DNA Marker); lane 1, dsRNA sample after treatment with both RNase-free DNase I and S1 nuclease. The AsFV1 band is indicated by a red arrow. (B) Graphical representation of the genome organization of AsFV1. The AsFV1 genome is 6828 nt in length and contains three ORFs (ORF1, ORF2, and ORF3). Open reading frames (ORF) are represented by boxes. The sizes of the encoded proteins and of the 5′ and 3′ untranslated regions (UTR) are indicated in amino acids and nucleotides, respectively. The conserved RNA-dependent RNA polymerase (RdRp) and RNA helicase (Hel) domains in the ORF1encoded protein are represented by gray and blue shading, respectively. (C) Phylogenetic tree based on the RdRp domains of AsFV1 and related viruses, constructed by the maximum-likelihood method with 1,000 bootstrap replicates in MEGA 7.0 software. Bootstrap values higher than 50% are shown. AsFV1 is indicated by a black star. including hypoviruses. All of the amino acid sequences clustered into groups corresponding to the proposed family "Fusariviridae" and the family Hypoviridae. AsFV1 was in the "Fusariviridae" cluster, and its closest neighbors in the tree were Pleospora typhicola fusarivirus 1, Plasmopara viticola lesion associated fusarivirus 1, and Plasmopara viticola lesion associated fusarivirus 3, all of which were separate from members of the genus Hypovirus (Fig. 1C) A multiple alignment and comparison of the RdRp domains of AsFV1 and other selected viruses showed that there were eight typical conserved motifs in the RdRp domains of fungal virus and viruses infecting lower eukaryotes (Fig. 2).
Sequence comparisons and phylogenetic analysis indicated that ASFV1 has all of the typical characteristics of a fusarivirus, suggesting that ASFV1 could be a new member of the proposed family "Fusariviridae".