Fusarivirus accessory helicases present an evolutionary link for viruses infecting plants and fungi

Abstract

A significant number of mycoviruses have been identified that are related to plant viruses, but their evolutionary relationships are largely unexplored. A fusarivirus, Rhizoctonia solani fusarivirus 4 (RsFV4), was identified in phytopathogenic fungus Rhizoctonia solani (R. solani) strain XY74 co-infected by an alphaendornavirus. RsFV4 had a genome of 10,833 ​nt (excluding the poly-A tail), and consisted of four non-overlapping open reading frames (ORFs). ORF1 encodes an 825 aa protein containing a conserved helicase domain (Hel1). ORF3 encodes 1550 aa protein with two conserved domains, namely an RNA-dependent RNA polymerase (RdRp) and another helicase (Hel2). The ORF2 and ORF4 likely encode two hypothetical proteins (520 and 542 aa) with unknown functions. The phylogenetic analysis based on Hel2 and RdRp suggest that RsFV4 was positioned within the fusarivirus group, but formed an independent branch with three previously reported fusariviruses of R. solani. Notably, the Hel1 and its relatives were phylogenetically closer to helicases of potyviruses and hypoviruses than fusariviruses, suggesting fusarivirus Hel1 formed an evolutionary link between these three virus groups. This finding provides evidence of the occurrence of a horizontal gene transfer or recombination event between mycoviruses and plant viruses or between mycoviruses. Our findings are likely to enhance the understanding of virus evolution and diversity.

Keywords: Fusarivirus; Helicase; Horizontal gene transfer (HGT); Hypovirus; Potyvirus.

Fig. 1

The molecular characterizations of Rhizoctonia solani fusarivirus 4 (RsFV4). A Colony morphology of strains XY74 and 190 on PDA medium. Pictures were taken after seven days of growth at 28 ​°C. B dsRNAs extraction and northern hybridization confirmation. The strain 190 is the RsFV4-free strain. Lane M: DNA molecular weight marker. The second lane contains XY74 dsRNA segments highlighted with yellow and red arrows that correspond to RsAEV1 and RsFV4, respectively. C Schematic genome organization of RsFV4. RsFV4 contains four non-overlapping ORFs (ORF1, ORF2, ORF3, and ORF4). The smaller ORF2 (520 aa) and ORF4 (542 aa) encode a putative protein of unknown function. The larger ORF3 (1550 aa) encodes a polyprotein of RNA dependent RNA polymerase (RdRp; pfam01699, E-value ​= ​3e-17) and HrpA-like RNA helicase (HrpA; cl34328, E-value ​= ​3e-07), while ORF1 encodes HrpA (cl34328, E-value ​= ​2.43e-08). D The distribution profile of transcriptomic reads and its maping against genome sequences of RsFV4. E Comparative genome organization of three R. solani fusariviruses and other representative fusariviruses. Schematic representation of the genome organizations and locations of each putative open reading frame. The conserved domains of helicase and RdRp were shown with different color frame. The members within the proposed family Fusariviridae have a complex and diverse genomic organization with two to four ORFs of different length. R. solani fusariviruses have two helicase domains, while other fusariviruses harbor a single helicase domain. RsFV4, Rhizoctonia solani fusarivirus 4; RsFV1, Rhizoctonia solani fusarivirus 1; RsFV2, Rhizoctonia solani fusarivirus 2; RnFV1, Rosellinia necatrix fusarivirus 1; FgFV1, Fusarium graminearum fusarivirus 1; SsFV1, Sclerotinia sclerotiorum fusarivirus 1.

Fig. 2

Multiple alignments of helicases in RsFV4 and its relatives. Alignment of two helicase domains of RsFV4 with their counterparts derived from viruses in the families Hypoviridae, Potyviridae, and the proposed family Fusariviridae. Seven motifs (I, Ia-VI) were detected in the sequence of the conserved helicase region. The default color scheme for ClustalW alignment in the Jalview program was used. Numbers within sequences correspond to the number of amino acid residues separating the motifs. Amino acid positions are indicated from the start and the stop regions of the ORF-encoded proteins.

Fig. 3

Phylogenetic analysis of the putative RsFV4 RdRp and helicases along with other related viruses. A Branch lengths are scaled to the expected underlying number of amino acid substitutions per site. The alignments were performed using the ClustalW algorithm, and the phylogenetic tree was constructed using the MEGAX program, by applying the maximum likelihood (ML) method with substitution model LG+G+I and 1000 bootstrap replications. The fusarivirus clade has been divided into five groups (I to V) and accession numbers follow virus names. Viruses within Hypoviridae and Potyviridiae are included as an outgroup. Bootstrap values (%) obtained with 1000 replicates are indicated on branches and branch lengths correspond to genetic distance; scale bar on the left corresponds to a genetic distance of 0.6. The Rhizoctonia solani fusarivirus 4 is highlighted in red color. B The phylogenetic tree was constructed using IQ-TREE with the best-fit model “Blosum62+F+R7.” Bootstrap values (%) obtained with 1000 replicates are indicated on branches and branch lengths correspond to genetic distance. The alignments were performed with MAFFT (Version 7.427) using the E-INS-i model and trimmed with trimAl. Selected viruses in families Hypoviridae, Potyviridae, and viruses that contain helicase belonging to superfamily II, were included in the phylogenetic tree. The RsFV4 is highlighted in red. Sequence information of all selected viruses has been provided in Supplementary Table S2.

Fig. 4

The impact of RsFV4 and RsAEV1 on R. solani. A Right plate: dual culture of R. solani strains XY74 and 190 on a PDA medium at 28 ​°C. Left plate: colony morphology of 190NV that is a new virus-infected isolates picked up from strain 190 side after dual-cultured (red arrow). Pictures were taken at 7 days post inoculations. B PCR detection of Rhizoctonia solani fusarivirus 4 (RsFV4), Rhizoctonia solani alphaendornavirus 1 (RsAEV1) using specific designed primers RsFV4-F1/R1 and RsAEV1-RsAEV1-F1/R1 (Supplementary Table S1). GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase) gene was used as a positive control for R. solani strains. C Pathogenicity assay of strains XY74, 190, and 190NV. Mycelium plugs of strains 190, XY74, and 190NV were inoculated on the detached rice leaves at 28 ​°C and continuously observed until 72 ​h post-inoculation (hpi). Pictures were taken each 24 ​h.