Characterization of a Novel Mitovirus Infecting Melanconiella theae Isolated From Tea Plants

A dsRNA segment was identified in the fungus Melanconiella theae isolated from tea plants. The complete dsRNA sequence, determined by random cloning together with RACE protocol, is 2,461 bp in length with an AU-rich content (62.37%) and comprises a single ORF of 2,265-nucleotides encoding an RNA-dependent RNA-polymerase (RdRp, 754 amino acids in size). The terminus sequences can fold into predicted stable stem-loop structures. A BLASTX and phylogenetic analysis revealed the dsRNA genome shows similarities with the RdRp sequences of mitoviruses, with the highest identity of 48% with those of grapevine-associated mitovirus 20 and Colletotrichum fructicola mitovirus 1. Our results reveal a novel member, tentatively named Melanconiella theae mitovirus 1 (MtMV1), belongs to the family Mitoviridae. MtMV1 is capsidless as examined by transmission electron microscope, efficiently transmitted through conidia as 100 conidium-generated colonies were analyzed, and easily eliminated by hyphal tipping method combined with green-leaf tea powder. MtMV1 has a genomic sequence obviously divergent from those of most members in the family Mitoviridae and some unique characteristics unreported in known members. This is the first report of a mycovirus infecting Melanconiella fungi to date.

Complementary DNA (cDNA) Cloning and Sequencing, and RT-PCR Amplication Purified dsRNA from WJB-5 was used as a template for cDNA synthesis following the previously described method (Xie et al., 2011) using a cDNA synthesis kit (Promega, Madison, WI, United States) and tagged random dN6 primer, 05RACE-3RT (Supplementary Table 1).The resulting cDNA was PCR-amplified using 2 × Gimmico GimiTaq PCR Mix (Gimmico Biotech Co., Wuhan, China) and 05RACE-3 primer (Supplementary Table 1).RT-PCR products were purified and then ligated to the pMD18-T vector (TaKaRa, Beijing, China), then transformed into competent cells of Escherichia coli DH5α.Positive clones with cDNA inserts were selected on Luria-Bertani (LB) agar medium containing ampicillin (50 µg/mL), checked with PCR using M13F-47/M13R-48 primers (Supplementary Table 1), and then submitted for sequencing.Specific primers, MtMV1-F and MtMV1-R (Supplementary Table 1) were designed for rapid amplification of cDNA ends (RACE) to amplify terminal sequences of the genome that were not cloned by the initial random cDNA synthesis.For RACE, the PC3-T7 loop adapter (Supplementary Table 1) was ligated to the 3 end of each strand in the purified dsRNA using T4 RNA ligase (TaKaRa, Beijing, China) at 16 • C for 18 h, as described previously (Ran et al., 2016).The oligonucleotide-ligated dsRNA was purified, denatured in DMSO, and subsequently reverse transcribed.RACE and nested PCR amplification were performed using sets of MtMV1-F/PC2 and PC2/MtMV1-R primers (Supplementary Table 1) to amplify the 3 -and 5 -termini respectively.PCR amplicons were purified, cloned into a pMD18-T vector, and sequenced.All amplicons were sequenced by Tsingke Biological Technology Co., LTD., Wuhan, China.Sequence homology searches were performed with the BLASTX search tool on the National Center for Biotechnology Information, NCBI2 .Sequence assemblies from the cDNA and the RACE clones were manipulated using DNAMAN software (version 7.0.2;Lynnon Corp., Vaudreuil-Dorion, QC, Canada) to obtain the complete genome sequence of the dsRNA extracted from the strain WJB-5.

Phylogenetic and Sequence Analyses
The viral ORF was identified using Expasy3 website, and the amino acid (aa) sequences encoded by the ORF were deduced.
The yeast mitochondrial genetic code was used for the translation of the ORF.The mycovirus sequence was searched using the NCBI BLASTX program to identify similar mycoviruses for phylogenetic analysis.Maximum-likelihood (ML) phylogenetic tree with 1,000 bootstrap replicates was constructed using DNAMAN software (version 7.0.2;Lynnon Corp., Vaudreuil-Dorion, QC, Canada) for the amino acid sequences of RdRp from WJB-5 dsRNA along with 26 mitoviruses and 4 narnaviruses retrieved from the GenBank database.The evolutionary distances were computed using the Poisson correction method (uniform rates between sites).The potential RNA secondary structures of the 5 -and 3 -terminal sequences were predicted, and the free energy ( G) was estimated using CLC genomics workbench software (version 21.0.4;QIAGEN Aarhus, Aarhus, Denmark) as described previously (Liu and Di, 2020).Mitoviral RdRp characteristic motifs were searched using the conserved domain database (CDD) search4 provided by NCBI, and multiple amino acid alignments.

Association of Strain WJB-5 With Viral-Like Particles
Mycelium plugs of the strain WJB-5 were grown on an autoclaved cellophane membrane placed on media for 8 days (d) at 25 • C. Minor modifications were adopted to isolate and purify viral  1.The bootstrap values were deduced based on 1,000 replicates and indicated beside the branches.The evolutionary distances were computed using the Poisson correction method and the branch lengths correspond to the genetic distance; the scale bar refers to the genetic distance of 0.05.A tringle indicates MtMV1.particles in the previously described method (Ran et al., 2016).In brief, 35 g mycelia of WJB-5 strain were harvested and crushed in the presence of liquid nitrogen.After that, the crushed mycelia were transferred into new sterile tubes containing 3 volumes of cold phosphate buffer (0.1 M sodium phosphate, pH 7.0, containing 0.2 M KCl and 0.5% β-mercaptoethanol) and gently mixed.The homogenate was centrifuged at 10,000 rpm and 4 • C for 15 min to remove the hyphal debris.After three ultracentrifugation cycles, virus particles were purified using a stepwise sucrose gradient of 20-50% (W/V).Following sucrose gradient ultracentrifugation, sucrose fractions were collected, diluted, and subjected to ultracentrifugation to pellet virus particles, and the pellet was resuspended again in 150 µL of phosphate buffer (0.1 mol/L sodium phosphate, pH 7.0).Viral dsRNA was recovered from purified viral particles by a phenolchloroform-based technique as described previously (Toni et al., 2018), fractionated by agarose gel (1%, w/v) electrophoresis and detected by UV transillumination.For negative staining, 10 µL purified suspension of the virus was loaded on a hydrophobic parafilm surface, and subsequently copper grids with a carbonformvar coating (200-mesh) were floated onto them for 3 min.After drying with a filter paper, the grids plated with virus particles were immediately refloated for 3 min in a drop of 2% (W/V) phosphotungstic acid solution.The excess suspension was removed using filter paper, and the grids were then air-dried for a few min.Finally, transmission electron microscopy (TEM) was used to visualize the samples.

Attempts to Cure Melanconiella Strains
To eliminate the mycovirus from WJB-5 and five other (WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, and WJB-1-20) strains of Melanconiella theae, hyphal tipping method together with thermal/dark treatment were conducted.All strains were separately incubated on PDA-T [potato dextrose agar medium containing 0.4% (w/v) green-leaf tea powder] (Yang and Zhang, 2019) media in the dark at 20 • C for 3 days.The hyphal tips in the colony margin were individually cut using a fine needle, transferred again to PDA-T in new Petri dishes (1 hyphal tip per dish), and incubated simultaneously.The colonies were subcultured on PDA-T at 20 • C in dark conditions for three consecutive subculturing.Finally, the subculturing was achieved on PDA through incubation at 20 • C and then used to detect MtMV1 dsRNA.To emphasize that the M. theae strains got cured from MtMV1, total RNA was extracted and RT-PCR amplification was performed using specific primers (MtMV1-F1: 5 -GTCTTATCCGTGTATTCTGGG-3 ; MtMV1-R1: 5 -GGCTTGAGGAACATTGAGA-3 ), as previously described.

Analysis of Mycovirus Transmission Through Conidia
Conidia of WJB-5 strain (harvested from a 30-d-old culture grown on PDA at 20 • C in the dark) were suspended in sterile distilled water to the final concentration of 3 × 10 7 conidia/mL.The resulting conidial suspension was poured on PDA plates and incubated at 20 • C in darkness, and used for culturing of singleconidium isolates.A total of 100 single-conidium isolates derived from WJB-5 were obtained and separately incubated on PDA plates at 20 • C for mycelial growth.After the colonies had grown, they were subjected to dsRNA extraction to detect the described virus infection.

Sequence and Molecular Characterization of WJB-5 dsRNA
The entire cDNA sequence of the dsRNA fragment of the strain WJB-5 was obtained through random priming cDNA synthesis, RT-PCR, and RACE cloning.Sequence assembly of thirty random cDNA clones revealed that the genome was composed of a single dsRNA segment with a molecular size of 2,461 bp and with an AU-rich content of 62.37%.Searching of the full-length genome and the deduced amino acid sequences using BLASTX and BLASTP, the coding protein shared 37.77-48.65%identities with the RdRp sequences of known mitoviruses belonging to the family Mitoviridae, and the highest identities are 48.65 and 48.35% with those of grapevine-associated mitovirus 20 (GaMV20) and Colletotrichum fructicola mitovirus 1 (CfMV1), respectively (Supplementary Table 2).Thus, the dsRNA component is proposed as a novel mitovirus and

Genomic Organization and Phylogenetic Analysis
Based on the yeast mitochondrial code, the genomic organization analysis revealed that the MtMV1 genome contains a single large ORF on the positive sense strand of the dsRNA (Figure 2A).
The ORF starts and terminates at nucleotide positions 46 and 2,310, respectively, of 2,265 bp in size, and potentially codes for an RdRp protein with 754 aa in size.The ORF contains multiple codons (10 UGA and 2 UGG) encoding Trp, and the UAG codon stops the translation.Moreover, the 5 -and 3 -untranslated regions (UTRs) are 45 and 151 bp long, respectively (Table 1).The phylogenetic analysis separated the known mitoviruses into two distinct clusters and placed MtMV1 together with CfMV1 and GaMV20 in cluster I, which is distantly separated from other members (Figure 2B).Based on the conserved domain database (CDD) search and multiple amino acid alignments, the six typical amino acid motifs (I-IV) characterized for the mitoviral RdRp domain superfamily were found to be conserved in MtMV1 (Figure 3).However, MtMV1 contains many unique amino acids as compared with the other members in the conserved motifs (e.g., positions 12 and 20 in motif I, 4, and 19 in motif II, 18 in motif III, 3 and 4 in motif IV), which were composed by threonines (Thr) for MtMV1 while leucines (Leu), isoleucines (Ile) or valines (Val) for the others (Figure 3).Moreover, an unique serine (Ser) in position 16 in motif IV and a unique histidine (His) in position 7 in motif V were observed for MtMV1, while alanine in motif IV and aspartic acids, asparagines or threonines in motif V in these positions for the others (Figure 3).

Predicted Secondary Structures of the Terminal Sequence
The 5 -and 3 -terminus sequences of the MtMV1 genome were subjected to the prediction of their secondary structures using the CLC genomic workbench software.The results showed that both 5 -and 3 -UTRs could be folded into potential stem-loop structures that stabilize the RNA genome, with G values of −14.3 and −56.5 kcal/mol, respectively ( Figures 4A,B).Additionally, the partial regions of both terminal sequences are reverse complementary, with positions 1 to 27 of 5 -UTR paired with positions 100 to 126 of 3 -UTR, and potentially fold into a panhandle structure with G values of −105 kcal/mol (Figure 4C).

Attempts to Check the Viral-Like Particles From WJB-5 Strain
To determine whether virions are associated with MtMV1, possible viral proteins were extracted from the mycelia of strain WJB-5 and subjected to ultracentrifugation in stepwise sucrose gradients.The sucrose fraction containing the MtMV1 dsRNAs was further purified and subjected to observation with a transmission electron microscope (TEM).No virus-like particles were observed under TEM, confirming that MtMV1 dsRNAs are naked like other mitoviruses.

Attempts to Cure WJB-5 Strain From Melanconiella Theae Mitovirus 1
To cure the MtMV1 from the WJB-5 strain and the other five strains of M. theae (WJB-1-1, WJB-1-2, WJB-1-9, WJB-1-12, and WJB-1-20), the hyphal tipping method combined with antiviral extracts (incorporating green-leaf tea powder in the culture medium) was used.After three sequential rounds of subculturing, MtMV1 was detected by dsRNA extraction.The results showed that MtMV1 was cured from all detected strains (Figure 5B), which was further confirmed by RT-PCR identification resulting in no target bands (1.98 kb in size) (Supplementary Figure 2).

Vertical Transmission of Melanconiella Theae Mitovirus 1 Through Conidia
To investigate whether MtMV1 can be vertically transmitted to the next generation through fungal conidia, conidial suspension prepared from the strain WJB-5 were smeared on PDA, and a total of 100 single-conidium generated colonies were subjected to dsRNA extraction.The results showed that MtMV1 was retained in all these subisolates, suggesting that MtMV1 has efficiently transmitted through the conidia of M. theae (Figure 5A).

Melanconiella theae Mitovirus 1 Confers No Obvious Effects on the Growth Rates and Morphologies of the Host Fungus
To access the effect of MtMV1 on the host fungus, six MtMV1infected and six MtMV1-free strains of Melanconiella spp.were cultured on PDA in darkness to access their growth rates and morphologies.Most of these strains had similar morphologies and growth rates, ranging from 4.4 to 6.1 mm/d for the MtMV1infected strains and 4.1 to 5.4 mm/d for the MtMV1-free ones, suggesting that no obvious effects were observed for MtMV1 related to the morphologies and growth rates of their host strains (Supplementary Figure 3).

DISCUSSION
A novel mycovirus, named MtMV1, was identified infecting the phytopathogenic fungus M. theae.MtMV1 shares most characteristics of members in the family Mitoviridae considering that mitoviruses have the simplest positive ssRNA genomes that harbor a single ORF encoding a putative viral RdRp for their replication (Cole et al., 2000;Hillman and Cai, 2013;Khalifa and Pearson, 2013), capsidless (Hillman and Cai, 2013), and their RdRps contain six conserved amino acid motifs (I-IV) (Osaki et al., 2015;Ran et al., 2016;Chen et al., 2017;Wang et al., 2019;Kocanová et al., 2020;Liu et al., 2021).Consequently, MtMV1 together with GaMV20 and CFMV1 were placed in cluster I as the third member in this group.
Since mitoviruses essentially replicate within mitochondria of their hosts (Polashock and Hillman, 1994;Cole et al., 2000;Osaki et al., 2005;Nibert, 2017;Fonseca et al., 2021), MtMV1 genome shares the most features of mitochondrial codons described before, e.g., the MtMV1 genome contains ten UGA codons, which encode Trp rather than function as a stop codon (Nibert, 2017;Vong et al., 2019), while only two UGG, and has rich of A-U content (62.37%) like other members (59.55 to 73.25%).Moreover, the initiation codon of MtMV1 ORF has a preference to be either A or U with a frequency of 68.3% in the third nucleotide position, similar to those of other members (Liu et al., 2019;Torres-Trenas and Pérez-Artés, 2020;Wang et al., 2021) and has a UAG stop codon, similar to the observation in other members with either UAA or UAG (Nibert, 2017).Additionally, the 5 -and 3 -terminus sequences of MtMV1 could be folded into stem-loop structures, and their reverse complementary regions allow to form a stable panhandle structure, which is also observed in other members (Hong et al., 1999;Khalifa and Pearson, 2013;Wang et al., 2021).Whereas, the size of MtMV1 3 -UTR (151 nt) is relatively longer than its 5 -UTR (45 nt), it is different as compared with other mitoviruses since their 3 -UTR sizes are all shorter than their 5 -UTR (ranging from 1 to 413 nucleotide longer) (Stielow et al., 2011;Xie and Ghabrial, 2012;Nibert et al., 2018).It is worth noting that MtMV1 has the smallest 5 -UTR in size compared with other members (ranging from 49 to 472 nt) in the family Mitoviridae whether some biological functions related to the unique traits of MtMV1 terminal sequences require further studies.Moreover, MtMV1 harbors a genomic sequence obviously divergent from those of most members in the family Mitoviridae as exemplified by having Thr-rich RdRp motifs as compared with other members (Figure 3).
MtMV1 is efficiently transmitted through all conidia of strain WJB-5 (100 conidium-generated colonies were checked) in agreement with those mitoviruses detected in a high percentage of the individual spore isolates, as exemplified by Fusarium circinatum mitovirus 1 (FcMV1) and Fusarium circinatum mitovirus 2-2 (FcMV2-2) that showed vertical transmission rates between 60 and 100% depending on the fungal isolate (Romeralo et al., 2018), however, both Fusarium verticillioides mitovirus 1 (FvMV1) and Fusarium andiyazi mitovirus 1 strain 162 (FaMV1-162) showed 100% vertical transmission rate through fungal conidia (Jacquat et al., 2020).In contrast, some mycoviruses are less efficiently transmitted through conidia, as exemplified by Colletotrichum camelliae filamentous virus 1 (CcFV-1) and Pestalotiopsis theae chrysovirus-1 (PtCV1) (Jia et al., 2017;Zhou et al., 2021).Generally, the efficient transmission of mitoviruses through asexual spores and sclerotia has hindered to understand mitovirus biology, partly due to lacking mitovirus-free subisolates used for infection, although a protoplast fusion-based protocol for horizontal transmission of a mitovirus has been developed (Shahi et al., 2019).In light of several reports of tea polyphenols exhibiting antiviral activities against various viruses, especially positive-sense single-stranded RNA viruses (Okada, 1978;Zhang et al., 2020;Mhatre et al., 2021), we tried the elimination of MtMV1 from the fungal host, it is worth noting that MtMV1 was easily cured by the hyphal tipping method combined with green-leaf tea powder in the culture medium as examined by dsRNA extraction and RT-PCR identification, which is expected to provide an efficient approach to obtain mitovirus-free host strains.Moreover, MtMV1 confers no obvious effects on the growth rates and morphologies as demonstrated by some homologous and isogenous strains.Thus, we did not proceed to viral transmission for more strict assessment of its biological effects.
Collectively, a novel mitovirus (MtMV1) from M. theae was characterized based on its taxonomic analysis, molecular characterization, genome organization, electron microscope observation, and some biological traits.MtMV1 harbors a genomic sequence obviously divergent with those of most members in the family Mitoviridae and some unique characteristics unreported in known members, contributing helpful information to understand the evolution, molecular and biological traits of mycoviruses.

FIGURE 2 |
FIGURE 2 | Genomic organization and phylogenetic analysis of MtMV1.(A) Genome organization of MtMV1.The start and end positions are labeled for the genome, untranslated regions (UTR), and the open reading frame (ORF).(B) ML phylogenetic tree was constructed using DNAMAN software based on RdRp sequences of MtMV1 and other members in the family Mitoviridae listed in Table1.The bootstrap values were deduced based on 1,000 replicates and indicated beside the branches.The evolutionary distances were computed using the Poisson correction method and the branch lengths correspond to the genetic distance; the scale bar refers to the genetic distance of 0.05.A tringle indicates MtMV1.

FIGURE 3 |
FIGURE 3 | Multiple alignments of the conserved motifs of RdRp in MtMV1 and other mitoviruses.Lines above the aligned sequences indicate to the positions of the motifs (I-VI).Numbers between every two motifs correspond to the number of aa residues separating the motifs.Identical, conserved, and semi-conserved aa residues are color-highlighted with homology level as 100% (black), 75% (magenta), 50% (turquoise), and 33% (yellow).

FIGURE 4 |
FIGURE 4 | Predicted secondary structures of the terminal sequences of MtMV1.(A,B) Stem-loop structures folded for the 5 -and 3 -UTR sequences of the genomic positive-strand, respectively.(C) Panhandle structure formed by the reverse complementary regions of the 5 -and 3 -UTRs ends of the MtMV1 genome.The CLC workbench software was used to predict the secondary structures of the terminal sequences and to calculate the free energy.The solid and open arrows indicate the 5 -and 3 -terminal sequences, respectively, in their initial and ending positions.
tentatively named Melanconiella theae mitovirus 1 (MtMV1).The complete nucleotide sequence of MtMV1 has been deposited in GenBank with the accession number MW802251.

TABLE 1 |
Comparison of genomic information of MtMV1 and other mitoviruses.