Molecular characterization of a novel fungal alphaflexivirus reveals potential inter-species horizontal gene transfer

Highlights

• •A +ssRNA virus, SsAFV2, was molecularly characterized in sclerotinia sclerotiorum.
• •SsAFV2 is a new member of the Botrexvirus genus within the alphaflexiviridae.
• •Phylogenetic analysis revealed the occurrence of potential inter-species HGT events.

Abstract

Sclerotinia sclerotiorum is a notorious phytopathogenic fungus that harbors diverse mycoviruses. A novel positive-sense single-stranded RNA virus, Sclerotinia sclerotiorum alphaflexivirus 2 (SsAFV2), was isolated from the hypovirulent strain 32–9 of S. sclerotiorum, and its complete genome was determined. The SsAFV2 genome contains 7,162 nucleotides (nt), excluding the poly (A) structure, and is composed of four open reading frames (ORF1–4). ORF1 encodes a polyprotein that contains three conserved domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). The ORF3 putative encodes coat proteins (CP), with ORF2 and ORF4 encoding hypothetical proteins of unknown functions. Phylogenetic analysis revealed that SsAFV2 clustered with Botrytis virus X (BVX) based on multiple alignments of helicase, RdRp, and CP, but the methyltransferase of SsAFV2 was most closely related to Sclerotinia sclerotiorum alphaflexivirus 1, suggesting that SsAFV2 is a new member of the Botrexvirus genus within the Alphaflexiviridae family, and also revealed the occurrence of potential inter-species horizontal gene transfer events within the Botrexvirus genus during the evolutionary process. Our results contribute to the current knowledge regarding the evolution and divergence of Botrexviruses.

Viruses infect virtually all forms of cells, and fungi are no exception. Knowledge of mycoviruses (fungal viruses) has expanded exponentially over the past 60 years, since the first definitive report of three viruses infecting the cultivated button mushroom, Agaricus bisporus (Hollings, 1962). Mycoviruses are widespread in all major fungal groups, including edible, medicinal, plant endophytic, and phytopathogenic fungi (Xie and Jiang, 2014). A few mycoviruses infecting phytopathogenic fungi can reduce the growth rate and/or virulence of their host, which has the potential to combat fungal diseases and supply materials to explore fungal biological mechanisms (Ghabrial et al., 2015). In addition, multiple infections by mycoviruses are common in fungi, which provides opportunities for studying virus-virus interactions as synergistic, neutral, or antagonistic (Hillman et al., 2018).

Members of Alphaflexiviridae of the order Tymovirales infect a wide range of hosts, from monocotyledonous to dicotyledonous plant species and filamentous fungi. Viruses belonging to the Alphaflexiviridae family were assigned to seven genera: Allexivirus, Botrexvirus, Lolavirus, Mandarivirus, Platypuvirus, Potexvirus, and Sclerodarnavirus. Their virions are flexuous filaments that are usually 12–13 nm in diameter and 470–800 nm in length, depending on the genus. Their viral capsid is composed of a single polypeptide ranging from 18 to 43 kDa in size, except for the genus Lolavirus, which has two capsid protein variants, and members of the genus Sclerodarnavirus, in which has no capsid proteins. The virions contain a single molecule of linear, positive-sense RNA of 5500–9000 nt, which is typically capped at the 5′-terminus with an m⁷G and has a polyadenylated tract at the 3′-terminus. There are five to seven genes, except for members of the genus Sclerodarnavirus, which have only one gene (Kreuze et al., 2020; Xie et al., 2006). The protein encoded by open reading frame 1 (ORF1) or ORF2 in members of the genus Platypuvirus, is homologous to the replication-associated proteins of the ‘alphavirus-like’ super group of RNA viruses (Martelli et al., 2007). This protein contains conserved methyltransferase, helicase, and RNA-directed RNA polymerase motifs (Batten et al., 2003). In all plant-infecting members of the family, except for members of the genus Platypuvirus, ORF 2–4 encode the ‘triple gene block’ proteins involved in the cell-to-cell movement (Verchot-Lubicz et al., 2010), and ORF5 encodes the viral capsid protein. So far, only four alphaflexiviruses that infect three plant pathogenic fungi, including Botrytis cinerea (Botrytis virus X, BVX), Botryosphaeria dothidea (Botryosphaeria dothidea botrexvirus 1, BdBV1), and Sclerotinia sclerotiorum (Sclerotinia sclerotiorum alphaflexivirus 1, SsAFV1; Sclerotinia sclerotiorum debilitation-associated RNA virus, SsDRV), have been discovered and characterized (Howitt et al., 2006; Xie et al., 2006; Yang et al., 2021; Ye et al., 2023).

S. sclerotiorum is an important necrotrophic phytopathogenic ascomycete fungus that can infect more than 700 plant species from more than 75 families, including important field crops, fruit trees, flowers, and many weeds (https://nt.ars-grin.gov/fungal databases/), and S. sclerotiorum causes significant economic losses. Recently, many novel mycoviruses have been characterized and identified in S. sclerotiorum (Jia et al., 2021; Mu et al., 2018; Zhang et al., 2019b). Eight reported mycoviruses in S. sclerotiorum belong to the order Tymovirales, three (Sclerotinia sclerotiorum deltaflexivirus 1, SsDFV1, SsDFV2, and SsDFV3) of which are positioned in the genus Deltaflexivirus within the family Deltaflexiviridea (Hamid et al., 2018; Li et al., 2016; Mu et al., 2021), two (Sclerotinia sclerotiorum debilitation-associated RNA virus; SsDRV and SsDRV2) are assigned to genus Sclerodarnavirus within family Alphaflexiviridae (Hu et al., 2014; Xie et al., 2006), two (Sclerotinia sclerotiorum mycotymovirus 1, and Sclerotinia sclerotiorum tymo-like RNA virus 4) are assigned to family Tymoviridae, and our group recently reported that SsAFV1 belongs to genus Botrexvirus within family Alphaflexiviridae (Ye et al., 2023). Here, we report the characterization of a second botrexvirus, Sclerotinia sclerotiorum alphaflexivirus 2 (SsAFV2), in the hypovirulent strain 32–9 of S. sclerotiorum.

The S. sclerotiorum strain 32–9 was originally isolated from a sclerotium collected from diseased rapeseed stalks in Sichuan Province, China, in 2016. The sclerotium surface was sterilized with 75% ethanol and washed thrice with autoclaved distilled water. A small portion of the sclerotium sample was cut and transferred onto potato dextrose agar (PDA; containing cephalosporin to a final concentration of 100 μg/mL) to cultivate at 20 °C. Four days later, the mycelia were transferred onto a fresh PDA medium and strain 32–9 was obtained. Strain 32–9 was stored in 20% glycerol water at 4 °C, and was cultured on the PDA to characterize its biological features. Compared with the virulent strain 1980, strain 32–9 had a slower growth rate, abnormal colony morphology with more aerial hyphae, and failed to produce sclerotia at the later growth stage (Fig. 1A). Although strain 32–9 normally produces and degrades oxalic acid (Fig. 1B, C), which is considered an important virulence factor in S. sclerotiorum (Liang et al., 2015), the lesions caused by strain 32–9 were significantly smaller than those induced by strain 1980 on rapeseed plant leaves (Fig. 1D, G). Similar to previously reported hypovirulent strains of S. sclerotiorum (Mu et al., 2021), strain 32–9 can be infected by one or more mycoviruses that are responsible for the hypovirulent features.

Fig. 1.

S. sclerotiorum strain 32–9 shows hypovirulent phenotypes. (A) Colony morphology of strains 1980 and 32–9. All strains were cultured on PDA plates for seven days prior to photography. (B, C) Oxalic acid secretion of strains 1980 and 32–9. All strains were cultured on 0.001% Bromophenol blue PDA plates for two days (B) and seven days (C) prior to photography. (D) Virulence assay of strains 1980 and 32–9 was carried out on the detached leaves of rapeseed. Photos and data were taken at 48 h post-inoculation (hpi). (E) RT-PCR confirmation of the assembled mycovirus-related contigs from strain 32–9 generated by Illumina sequencing. The primers were designed according to the sequences of the contigs; primers and the predicted sizes of amplicons are listed in Table S1. SsActin, the internal control gene, actin, of S. sclerotiorum. Lane M, DNA marker 2000 bp (TaKaRa, Dalian, China). (F) Growth rate of strains 1980 and 32–9 at 20°C. (G) Lesion diameters induced by the two strains on detached rapeseed leaves 20°C at 48 hpi. Error bars indicate the SD from six sample means. The different letters on the top of each column indicate significant differences at the P < 0.05 level of confidence, according to the t-test.

To identify the mycoviruses infecting strain 32–9, total RNA was extracted using an RNAisoKit according to the manufacturer's instructions (Takara, Dalian, China). The extracted total RNA was electrophoretically assessed on a 1% agarose gel for 20 min at 120 V using 0.5 × TBE and stained with ethidium bromide, after which the gel was photographed using a gel imager (Bio-Rad). High-quality RNA was used for metatranscriptome sequencing, and virus-related contigs were extracted as previously described (Jia et al., 2021). Specific primers were designed based on the viral contigs obtained to confirm the presence of mycoviruses in strain 32–9 (Fig. 1E, Table S1). Strain 32–9 was finally confirmed to be coinfected with two mycoviruses, SsAFV2 and SsOLV22 (Sclerotinia sclerotiorum ourmia-like virus 22, OK165499). The obtained genome of SsOLV22 is 3, 516 nt long and has a 96.7% nucleotide identity to the previously reported SsOLV22 infecting strain HC025 of S. sclerotiorum (Wang et al., 2022). We recorded the two newly detected viral sequences in the GenBank database with the accession numbers OQ865609 for SsAFV2 and OQ869265 for SsOLV22. In this study, we focused on the characterization of SsAFV2.

To confirm the accuracy of the sequence of SsAFV2 obtained using high-throughput sequencing technology, the extracted high-quality RNA of strain 32–9 was used as a template for first-strand cDNA synthesis by real-time reverse transcription polymerase chain reaction (rRT-PCR) with Moloney murine leukemia virus (M-MLV) reverse transcriptase (TaKaRa, Dalian, China) and random dN6 primers (5′-CGATCGATCATGATGCAATGCNNNNNN-3′). Subsequently, a series of PCRs were conducted using four SsAFV2-specific primers to produce four overlapping sequences. To obtain the terminal sequence of SsAFV2, the adaptor PC3-T7 loop (5′-p-GGATCCCGGGAATTCGGTAATACGACTCACTATATTTTTATAGTGAGTCGTATTA-OH-3′) was ligated to the genomic RNA of SsAFV2 in the presence of T4 RNA ligase as previously described (Li et al., 2016), followed by reverse transcription. The terminal cDNA sequence was amplified using primer PC2 (5′-p-CCGAATTCCCGGGATCC-3′), a complementary primer to the adapter of the PC3-T7 loop, and SsAFV2-specific primers corresponding to the 5′- and 3′-terminal sequences (Table S1). All PCR products were separated using agarose gel electrophoresis, recovered using a gel extraction and purification kit (Axygen, NY, USA), and cloned into the pMD18-T vector (TaKaRa, Dalian, China). M13 primers (Table S1) were used for the PCR detection of positive clones. Each PCR and sequencing reaction was independently repeated thrice to ensure sequence accuracy.

We assembled and verified the complete genomic sequence of SsAFV2 based on an integrative analysis of metatranscriptome data, RACE, and next-generation sequencing (Fig. 2A). The complete genome of SsAFV2 was found to be 7162 nucleotides (nt) in length with a GC content of 54.26% (21.43% G and 32.83% C). SsAFV2 contains ORF1–4 with a 5′-untranslated region (5′-UTR; 128 nt) and a 3′-UTR (151 nt). SsAFV2 has a polyadenylated structure (37 nt in length) at the 3′ terminus, like other members of the Alphaflexiviridae family (Howitt et al., 2006; Yang et al., 2021; Ye et al., 2023). ORF1 putatively encodes a polyprotein (162.18 kDa) that contains three conserved domains: methyltransferase, helicase, and RNA-dependent RNA polymerase (RdRp). The ORF2 and ORF4, encode a hypothetical protein of 39.21 kDa and 15.11 kDa, respectively. Database searches of the nucleotide and amino acid sequences of ORF2 and ORF4 did not reveal any significant homology with other known nucleotide and protein sequences. The ORF3 encodes a hypothetical protein of 31.55 kDa; BLASTp searches with ORF3 showed significant identity to the coat proteins of ‘potex-like’ viruses, such as BVX (NP_932,309.1, 45.69%), SsAFV1 (UCR95344.1, 36.63%), BdBV1 (QQD86175.1, 42.93%), Ferula potexvirus 1 (QQG34579.1, 38.95%) and Donkey orchid symptomless virus (AKH39767.1, 43.87%). A MOTIF (MOTIF: http://www.genome.jp/tools/motif/) search of SsAFV2’s ORF3 revealed that it contained a Flexi_CP superfamily protein domain (pfam00286, E-value=8e-38), which is commonly found in potex- and carlaviruses (Majumder and Baranwal, 2011). Multiple alignments of the ORF3-encoded protein with other members of Alphaflexiviridae family showed that SsAFV2 has a putative salt bridge (Fig. 2B), which has been reported to be the hydrophobic core in flexuous, rod-shaped ssRNA viruses in plants (Dolja et al., 1991; Verchot-Lubicz et al., 2010).

Fig. 2.

Genomic properties of SsAFV2. (A) Schematic representation of the genomic organization of SsAFV2 and other related mycoviruses. Different proteins are represented by different colored boxes, MTR, Methyltransferases; HEL, Helicase; RdRp, RNA-dependent RNA polymerases; CP, coat proteins. (B) Multiple alignment of the CP of SsAFV2 and other flexuous rod-shaped viruses are potentially involved in salt-bridge formation. The conserved positively (Arg) and negatively (Asp) charged residues are indicated by red boxes. (C) Multiple alignment of the region corresponding to the RdRp domain of SsAFV2 and other selected alphaflexiviruses. Eight core RdRp motifs are indicated with the lines above. The starting amino acid position and spacing between motifs are indicated. Multiple alignment of the selected viral RdRPs was performed using the MAFFT online version 3.0 tool.

Multiple sequence alignments were performed using the amino acid sequence of the conserved RdRp domain of SsAFV2 and the corresponding sequences of representative viruses from the family Alphaflexiviridae. RdRp encoded by SsAFV2 shares highly conserved domains with closely related viruses and contains eight conserved domains of RNA viral replicase, including a putative "GDD" motif (Fig. 2C). The conserved motifs of RdRp, methyltransferase, helicase, and CP protein sequences from SsAFV2 and some representative viruses in the six genera of the family Alphaflexiviridae, as well as some members of the families Betaflexiviridae, Deltaflexiviridae, Gammaflexiviridae, and Tymoviridae, were used to construct a phylogenetic tree. Multiple sequence alignments of the selected viral proteins were performed using MAFFT online version 3.0 (Nakamura et al., 2018). The tree was constructed using the PhyML online web server based on MAFFT alignment with 1000 bootstrap replicates. The resulting phylogenetic tree was exported using iTOL (https://itol.embl.de/) and Adobe Illustrator 2021. The phylogenetic tree of RdRp, CP, and helicase revealed that SsAFV2 clustered with BVX, but that the methyltransferase of SsAFV2 phylogenetically forms an independent clade with that of SsAFV1 (Fig. 3, Fig. 4), which suggests an inter-species horizontal gene transfer event within the genus Botrexvirus. In addition, the genomic structure of SsAFV2 has significant difference from known members of Botrexvirus (Fig. 2A).

Fig. 3.

Phylogenetic tree of SsAFV2 based on RdRp. A maximum likelihood phylogenetic tree was constructed based on multiple alignment of virus replicases. Viruses belonging to different families of Tymovirales were selected. The tree was constructed using the PhyML online web server based on MAFFT alignment with 1000 bootstrap replicates. Bootstrap values (%) obtained with 1000 replicates are indicated on the branches, and branch lengths correspond to genetic distance; the scale bar at the lower left corresponds to genetic distance. The resulting phylogenetic tree was exported using iTOL and Adobe Illustrator 2021 software. SsAFV2 is shown in red. SsDRV1 & 2, Sclerotinia sclerotiorum debilitation-associated RNA virus 1& 2. Green dots represent viruses that infect plants, and black dots represent viruses that infect fungi. The triangles represent the phylogenetical groups containing viruses that belong to the same genus.

Fig. 4.

Two maximum likelihood phylogenetic tree of SsAFV2 were constructed based on methyltransferases (A) and helicases (B). Phylogenetic trees were constructed as described in the legend of Fig. 3. The triangles represent the phylogenetical groups of viruses belong to same family.

Unexpectedly, we found that the nucleotide sequence “GAAAAC” did not immediately follow the cap, which is typical of the specific cap/terminal sequence combination present in members of the plant virus genus, Potexvirus (Howitt et al., 2006). In addition, although ORF3-encoded protein could predict alphavirus CP in SsAFV2 (Fig. S1), virions could not be isolated by sucrose or cesium chloride gradient centrifugation.

To eliminate the mycovirus, we prepared protoplasts of strain 39–2 as previously reported methods (Zhang et al., 2009) and finally obtained 48 protoplast-regenerated isolates. Only three isolates (R9, R14, and R17) were infected by SsAFV2, indicating that SsAFV2 was easily eliminated by protoplast isolation with 93.75% of elimination rate. R17 harbored SsAFV2 alone, while R48 was infected by SsOLV22 alone (Fig 5A). However, virus-free strains failed to produce sclerotium on PDA (Fig. 5B). Virulence assay suggested that isolates R17 and R48 caused similar lesion to strain 32–9, but form significantly smaller lesion than virus-free isolates (R4 and R5) on the detached leaves of tomato (Fig. 5C, D), suggesting that SsAFV2 and SsOLV22 were associated to hypovirulence on S. sclerotiorum.

Fig. 5.

SsOLV22 and SsAFV2 show hypovirulent phenotypes. (A) RT-PCR confirmation of mycovirus content in strain 32–9 and its protoplast-regenerated isolates. The primers were designed according to the viral sequences; primers and the predicted sizes of amplicons are listed in Table S1. SsActin, the internal control gene, actin, of S. sclerotiorum. Lane M: DNA marker 2000 bp (TaKaRa, Dalian, China). Colony morphology (B) and virulence assay (C) of strain 32–9 and its protoplast-regenerated isolates. All isolates were cultured on PDA plates for seven days prior to photography and virulence assay was carried out on the detached tomato leaves (20 °C, 48 hpi). (D) Lesion diameters induced by S. sclerotium from Fig. 5C. Error bars indicate the SD from six sample means. The different letters on the top of each column indicate significant differences at the P < 0.05 level of confidence according to the t-test.

In conclusion, this study demonstrated that SsAFV2 related to hypovirulence is a novel member of the Botrexvirus genus in the Alphaflexiviridae family, and revealed that potential inter-species horizontal gene transfer has occurred.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32072475), the National Key Research and Development Program of China (2022YFA1304400), the Fundamental Research Funds for the Central Universities (2021ZKPY005), and the Earmarked Fund for CARS-12.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

• Data will be made available on request.