A special satellite-like RNA of a novel hypovirus from Pestalotiopsis fici broadens the definition of fungal satellite

Satellites associated with plant or animal viruses have been largely detected and characterized, while those from mycoviruses together with their roles remain far less determined. Three dsRNA segments (dsRNA 1 to 3 termed according to their decreasing sizes) were identified in a strain of phytopathogenic fungus Pestalotiopsis fici AH1-1 isolated from a tea leaf. The complete sequences of dsRNAs 1 to 3, with the sizes of 10316, 5511, and 631 bp, were determined by random cloning together with a RACE protocol. Sequence analyses support that dsRNA1 is a genome of a novel hypovirus belonging to genus Alphahypovirus of the family Hypoviridae, tentatively named Pestalotiopsis fici hypovirus 1 (PfHV1); dsRNA2 is a defective RNA (D-RNA) generating from dsRNA1 with septal deletions; and dsRNA3 is the satellite component of PfHV1 since it could be co-precipitated with other dsRNA components in the same sucrose fraction by ultra-centrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs. Moreover, dsRNA3 shares an identical stretch (170 bp) with dsRNAs 1 and 2 at their 5′ termini and the remaining are heterogenous, which is distinct from a typical satellite that generally has very little or no sequence similarity with helper viruses. More importantly, dsRNA3 lacks a substantial open reading frame (ORF) and a poly (A) tail, which is unlike the known satellite RNAs of hypoviruses, as well as unlike those in association with Totiviridae and Partitiviridae since the latters are encapsidated in coat proteins. As up-regulated expression of RNA3, dsRNA1 was significantly down-regulated, suggesting that dsRNA3 negatively regulates the expression of dsRNA1, whereas dsRNAs 1 to 3 have no obvious impact on the biological traits of the host fungus including morphologies and virulence. This study indicates that PfHV1 dsRNA3 is a special type of satellite-like nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein, which broadens the definition of fungal satellite.

Satellites are subviral agents which lack genes that could encode functions needed for replication, and depend on the co-infection of a host cell with a helper virus, which consist of satellite viruses and satellite nucleic acids, discriminating by the presence of a structural protein [13]. Satellite nucleic acids include ssDNAs and ssRNAs, associated with plant or animal viruses, and double stranded RNAs (dsRNAs), are mainly associated with mycoviruses, namely the families Totiviridae and Partitiviridae. Satellites do not constitute a homogeneous taxonomic group, and have no taxonomic correlation with their helper viruses, and have arisen independently a number of times during virus evolution [14]. Since satellites were mostly characterized as ssRNA satellites that use ssRNA plant viruses as helpers, and those associated with mycoviruses remain rarely characterized.
Pestalotiopsis spp. belong to the family Amphisphaeriaceae that are distributed widely throughout tropical and temperate regions. They are common phytopathogens reducing production and causing enormous losses in either horticultural plants or forest trees [15]. For example, in tea plants, P. theae and P. longiseta are responsible for tea gray blight disease, which leads to large brown spots with the formation of apparent concentric rings in the late period of disease on mature and old leaves of tea plants, and even leads to death of tender branches of infected tea seedlings; P. theae, P. camelliae, and P. clavispora were also identified in association with small brown-black spots on tea tender leaves [16,17]. However, endophytic Pestalotiopsis species can confer fitness to the host plants, and are also thought to be a rich source for bioprospecting compared to other fungal genera since they produce novel compounds with medicinal, agricultural and industrial applications [17]. Therefore, Pestalotiopsis fungi live in host plants with a complex lifestyle, being parasitic, commensal, or mutualistic. Mycoviruses might have participated in the transition of these lifestyles, since a chrysovirus, the first mycovirus isolated from Pestalotiopsis fungi, can convert its host fungus from a phytopathogenetic one to a nonpathogenetic endophyte on tea leaves, conferring high resistance to the host plants against the virulent P. theae strains [15]. It is fascinating to characterize more mycoviruses to understand the virus taxonomy, evolution, molecular and biological traits related to this important fungal species. Specifically, characterization of a hypovirus in this fungal species remains a great interesting since hypoviruses represent the classic and successful examples for biological control of plant diseases [18].
In this study, a novel special satellite RNA associated with a novel hypovirus from P. fici with three components (RNAs 1 to 3) was identified and characterized, which represent a novel class of satellite nucleic acids, illustrating some novel molecular and biological traits of a fungal satellite.

Fungal strains and cultures
P. fici strain AH1-1 was isolated from a tea leaf showing the typical symptoms of tea grey blight disease collected in Liu'an city, Anhui province, China, and was identified based on morphologies and multi-loci sequences as previously described [19], and strain AH1-1V -, AH1-1-14 and AH1-1-24 are subisolates generated from single conidia of strain AH1-1. Strain AH1-1Vis a cured subisolate of AH1-1 by elimination of dsRNA components, and conferred hygromycin (hyg)-resistant ability by transformation of a hyg gene to generate strain AH1-1 hyg V -. P. theae TP-2-2W, JWX-3-1, and CJB-4-1 were collected from tea leaves in Yichang city, Hubei province, China, and were identified on their morphologies and internally transcribed spacer (ITS) sequence. P. theae strain LI41 was isolated from a healthy tea leaf collected in Xuan'en county, En'shi prefecture, Hubei province, China, and LI41-1P1 is a subisolate of P. theae LI41, both of which were infected by Pestalotiopsis theae chrysovirus-1 (PtCV1) [15]. All strains were grown at 25˚C in the dark for 3-5 days on potato dextrose agar (PDA; 20% diced potatoes, 2% glucose, and 1.5% agar) unless otherwise stated.

dsRNA extraction, purification, and enzymatic treatments
For dsRNA extraction, fungal mycelial plugs were inoculated onto sterilized cellophane disks on PDA plates at 25˚C in the dark for 4 to 5 days, and the mycelia were collected and subjected to dsRNA extraction as previously described [20]. The resulting nucleic acids were treated with 2 U DNase I (New England Biolabs), 10 U S1 nuclease (Thermo Scientific) at 37˚C for 1 h, or those without treatments were fractionated by electrophoresis on 1.2% agarose gels with Tris-acetate-EDTA (TAE) buffer and visualized by staining with ethidium bromide. Each dsRNA band was excised, purified using a gel extraction kit (Qiagen), dissolved in DEPCtreated water and stored at −80˚C until use.

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A special satellite-like RNA from polymerase chain reaction (PCR) products were treated with S1 nuclease or/and DNase I as indicated above, and with 200 ng/mL RNase A (Thermo Scientific) in 2×saline sodium citrate (SSC) (300 mM NaCl, 30 mM sodium citrate, pH 7.0) or 0.1×SSC as previously described [20].

Cloning, sequencing, and sequence analysis
The sequences of the dsRNAs were determined by cloning and sequencing amplicons generated by reverse transcription and polymerase chain reaction (RT-PCR) using the random primers 05RACE-3RT and 05RACE-3 (S1 Table) as previously described [19]. The 5 0 -and 3 0terminal sequences of the dsRNAs were obtained by cloning and sequencing the RT-PCR amplicons generated using a standard RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) protocol, including the usage of PC3-T7loop and PC2 (S1 Table). The oligonucleotide primers used for RLM-RACE were designed based on sequence information obtained from the randomly primed amplicons [22]. At least three independent clones of each amplicon were sequenced in both directions, by Sangon Biotech Co., Ltd, Shanghai, China. Sequence similarity searches were performed using the BLASTn program for nucleic acids or BLASTp for putative proteins against the National Center for Biotechnology Information (NCBI) databases. Multiple alignments of nucleic and amino acid (aa) sequences were conducted using MEGA7 and MAFFT online (MAFFT alignment and NJ / UPGMA phylogeny (cbrc.jp)). The phylogenetic tree for RNA-dependent RNA polymerase (RdRp) sequences was constructed using MEGA 11 with the Maximum Likelihood method [23]. Open reading frames (ORFs) were deduced using an ORF finder (https://www.ncbi.nlm.nih.gov/orffinder/). Conserved domains were predicted using Phyre2 (https://www.sbg.bio.ic.ac.uk/phyre2). Internal ribosomal entry site (IRES) elements were predicted by aligning with IRESite database online (IRESite: The database of experimentally verified IRES structures) [24].
Co-precipitate analysis of dsRNAs was conducted by sucrose gradient centrifugation according to the methods used for the purification of viral particles previously described [15]. The resulted aliquots of each fraction (100 μL) were subjected to dsRNA extraction to monitor for the presence of viral dsRNAs.
RNase A treatment was conducted as previously described [26]. Briefly, fungal mycelia were cultured on cellophane-PDA for 3 days at room temperature and powdered in the presence of liquid nitrogen. The mycelial powder was homogenized in 2 volumes (vol/wt) of 0.1 M sodium phosphate (pH 7.2) and centrifugated at 10000 r/min for 10 min at 4˚C. The supernatant was treated with RNase A (10 μg/mL) (Thermo scientific, Shanghai) for 30 min at 37˚C. The solution was extracted with phenol and chloroform to inactivate RNase A, followed by chloroform extraction.

Engineering of RNA3-over-expressed strain and quantitative real-time PCR (qRT-PCR) analysis
To generate RNA3-over-expressed strains, RNA3 cDNAs were inserted into the multi-cloning sites of vector pCAMBgfp after linearization by Pst I [27]. The constructed vector was then transformed to the strain AH1-1-24 via PEG4000 mediated protoplasts transformation as previously described [15].
The total RNAs from mycelia were extracted by Trizol agent and subjected to digestion by 5×gDNA digesture (Foregene, Chengdu, China) to remove the genomic DNAs, followed by cDNA synthesis using 2× reverse transcriptase (Foregene, Chengdu, China) and amplification using 2×TSINGKE master qPCR mix (Tsingke, Beijing, China) using specific primers designed for quantitative analysis in CFX96 Real-Time PCR Detection System (Bio-Rad, USA). Amplification was conducted for 40 cycles of 95˚C for 10 s, 56˚C for 10 s and 72˚C for 15 s after initial heating at 95˚C for 1 min. A partial fragment of the Actin gene was used to normalize the RNA samples for each qRT-PCR, and each treatment was conducted with three technical replicates.

Contact cultures of P. fici isolates
Horizontal transmission of the virus was conducted as previously described [20]. Briefly, both donor and recipient strains were cultured together on the same Petri dishes at 25˚C for 7 days, and allowed to physically contact each other. Following contact, mycelial agar plugs were excised from the contact area of two strains and sub-cultured onto fresh PDA plates. Seven independent donor-recipient pairs were assessed, and two mycelial agar plugs were selected from each pair for further analysis, resulting in a total of 14 isolates.

Growth rate, morphology, and virulence assays
Fungal growth rates and morphologies were assessed as previously described [15]. Three biological replicates for each strain were monitored and the resulting data were subjected to statistical analysis as described below. Fungal virulence was determined following inoculation of detached tea leaves (Camellia sinensis, C. sinensis vars. Echa no.1 or Fuyun no.6) as previously described [15]. At 4 day past inoculation (dpi), lesions that developed on the inoculated leaves were measured. Eight biological replicates for each strain were monitored and the resulting data were subjected to statistical analysis as described below.

Statistical analyses
The biological data were statistical analysis using SPSS Statistics 17.0 with one-way analysis of variance (ANOVA) and Tukey post-hoc tests (Win Wrap Basic; http://www.winwrap.com). The mean values for the biological replicates are presented as column charts with error bars representing standard error of mean (SEM). The graphs were produced in Excel (Microsoft) and GraphPad Prism 7 (GraphPad software). P-values < 0.05 were considered to indicate statistical significance, while P-values < 0.01, extremely significant.

A complex of dsRNA segments were extracted from P. fici strain AH1-1
Nucleic acid preparations enriched in dsRNA were obtained from P. fici strains AH1-1 and TP-2-2W isolated from tea leaves collected in China, and were subjected to digestion with DNase I and S1 nuclease and then agarose gel electrophoresis. The results showed that three dsRNAs (nominated 1-3 according to their decreasing sizes) were detected in preparations of

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A special satellite-like RNA strain AH1-1 but not in a control strain TP-2-2W ( Fig 1B). Of these components, dsRNA1 migrated together with the fungal genomic DNAs as electrophoresis on an agarose gel, and became apparent after treatment by DNase I (Fig 1B). The sequences of the full-length cDNAs

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A special satellite-like RNA of dsRNAs 1-3 were determined by assembling partial-length cDNAs that were amplified from the purified dsRNAs using RT-PCR and RLM-RACE protocols. The corresponding sequences were deposited in GenBank with accession numbers OP441373-OP441375.
The dsRNA nature of the three observed bands was further assessed by treatments with DNase I, S1 nuclease or RNase A (in 2× and 0.1×SSC), together with an ssRNA control (in vitro dimeric transcripts of peach latent mosaic viroid (PLMVd) and dsRNAs from a dsRNA mycovirus (Botryosphaeria dothidea RNA virus 1, BdRV 1). The RNAs extracted from strain AH1-1 together with BdRV 1 dsRNAs were digested by RNase A in 0.1×SSC, but they resisted digestion by DNase I, S1 nuclease, and RNase A in 2×SSC. In sharp contrast, PLMVd transcripts were completely degraded by S1 nuclease and by RNase A under both ionic conditions, but resisted digestion by DNase I; the genomic DNAs (extracted together with BdRV 1) and the cDNA plasmid of PLMVd (used for in vitro transcription) were completely degraded by DNase I while resistant to RNase A and S1 nuclease ( Fig 1C). These data strongly support that the nucleic acids extracted from strain AH1-1 were indeed dsRNAs instead of DNAs or ssRNAs.

dsRNA 1 composes the genome of a novel hypovirus
The complete nucleotide sequence of dsRNA1 is 10316 base pair (bp) in length, consisting of a single putative ORF beginning at AUG (nt positions 513-515) and terminating at UAG (9600-9602), coding for a polyprotein of 3029 aa with an approximate molecular mass of 345.9 kDa.
The deduced polyprotein aa sequence contains conserved domains of RNA dependent RNA polymerase (RdRp), viral RNA Helicase (Hel), and a most likely papain-like protease (Pro) ( Fig  2B). Within the RdRp domain, nine conserved motifs (Ia-VII), characteristic for an RNA virus [28], were detected (Fig 3A), and seven conserved motifs (I-VI) relatively conserved in (+) ssRNA were detected within the Hel domain ( Fig 3C); while in the possible Pro domain, only cysteine and glycine were detected whereas the core residue histidine was absent (Fig 3B). The 5 0 -untranslated region (UTR) of dsRNA1 is 512 nts in size, contains six AUG codons (S1C Fig), and the 3 0 -UTR is 714 nts including a 6 nt-sized adenine tail (poly A) (Fig 2B). Each UTR region forms a compact and complex stem-loop structure as predicted in RNAfold (S1A and S1B Fig).

dsRNA2 is a D-RNA of PfHV1
The complete nucleotide sequence of dsRNA2 is 5511 bp in length excluding the poly (A) tail, consisting of a single putative ORF (nt positions 510-4043), coding for a polyprotein of 1177 aa with an approximate molecular mass of 133.5 kDa. Alignment with dsRNA1 indicated that the total sequence of dsRNA2 is separately identical with dsRNA1 in five regions, i.e., nt position 4 to 3939, 5988 to 6225, 6220 to 6508, 6512 to 6770, and 6774 to 7562. Namely, dsRNA2 is a D-RNA generated from dsRNA1 by deletion three regions from nts 1 to 3, 3940 to 5987 and 7563 to 10316 (Fig 4A). Moreover, an inversion was observed in dsRNA2 sequence between nt position 4175 to 4433 and 4434 to 4722 as compared with dsRNA1 ( Fig 4A).

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A special satellite-like RNA

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A special satellite-like RNA To further confirm that dsRNA2 as a D-RNA generated from dsRNA1, a digoxigenin (DIG)-labeled riboprobe (termed riboprobe 1) binding to the nt 1588 to 1934 of dsRNA1 was synthesized in vitro transcription and hybridization with the nucleic acid preparations of P. fici strain AH1-1. As expected, two hybridized signals were observed corresponding to the electrophoresis position of dsRNAs 1 and 2, further supporting the D-RNA nature of dsRNA2.

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A special satellite-like RNA To further check whether PfHV1 dsRNAs are encapsidated, nucleic acid preparations of P. fici strain AH1-1 without protein-denaturing treatment process were subjected to RNase A treatment at 37˚C or on ice, and P. theae strain LI41-1P1 infected by PtCV1 (an encapsidated dsRNA mycovirus) was involved in parallel as a control. The results showed that PfHV1 dsRNAs, similar to PtCV1, are resistant to RNase A treatment, suggesting that they are encapsulated (Fig 4C).

dsRNA3 is a satellite-like RNA (SatL-RNA) of PfHV1
The complete nucleotide sequence of dsRNA3 is 631 bp in length without a poly (A) tail, and it has a small putative ORF (nt positions 248-400) on its positive strand coding for a protein of 50 aa with an approximate molecular mass of 5.75 kDa. dsRNA3 contains nine putative IRES elements in nt positions 1-247 (e-values 2.8-31) as aligned with those deposited in IRESite database (S3 Table). Alignment of dsRNA3 with dsRNAs 1 and 2 indicated that their first 170 bp were 100% identical, excluding 3 nts deletion at 5 0 -termini of dsRNA 2, while the remaining sequence had no detectable similarity with both dsRNAs. To further confirm that dsRNA3 is identical with dsRNAs 1 and 2 at the 5 0 -termini while heterogenetic in the remaining, two DIG-labeled riboprobes (termed riboprobe 2 and 3) binding to the identical and heterogenetic regions of dsRNA1, respectively, were synthesized in vitro transcription and hybridization with the nucleic acid preparations of P. fici strain AH1-1. As riboprobe 2 was utilized, three hybridized signals were observed corresponding to the electrophoresis position of dsRNAs 1 to 3; while as riboprobe 3 was utilized, only one hybridization signal was observed corresponding to the electrophoresis position of dsRNA3 ( Fig 4B). Thus, the hybridization analysis supports the conclusion from the sequence alignment.
To further confirm that dsNRA3 is a SatL-RNA hosted by PfHV1, the presumed viral vesicles were purified by centrifugation in stepwise sucrose gradients (10-35% with 5% sucrose increments), and subjected to nucleic acids extraction. Agarose gel electrophoresis of the resulted nucleic acids showed that dsRNA3 together with the typical pattern of PfHV1 dsRNAs 1 and 2 was most recovered from the 15% fractions ( Fig 4D). These data suggest that dsRNAs 1 to 3 are encapsulated together, and it supports that dsNRA3 is a SatL-RNA of PfHV1.

dsRNA3 negatively regulates the replication of PfHV1
To investigate whether the dsRNA3 affects the replication of PfHV1, the full-length of dsRNA3 cDNAs was engineered into an expression vector containing a hygromycin resistance marker, and transformed into the protoplasts prepared from the dsRNA1-only infected strain AH1-1-24 of P. fici. With the resistance selection and qPCR identification, three derivative subisolates, termed AH1-1-24 SatL-OE1 to AH1-1-24 SatL-OE3 , transformed by RNA3-expression vector were obtained. Expression analysis of dsRNA1 together with RNA3 using qRT-PCR in the three resulting subisolates revealed that the expression levels of RNA3 were significantly up-regulated up to 1.96 to 9.72 folds in these transformed subisolates (Fig 4E), whereas dsRNA1 were significantly down-regulated, up to 30.03% to 70.63% (Fig 4E). These results suggest that the overexpression of the satellite-like RNA inhibits the replication of PfHV1. Whereas, no obvious accumulation of SatL-RNA was observed in these over-expressed strains as checked by electrophoresis analysis of their dsRNAs on agarose gel (S2A Fig).

PfHV1 dsRNAs are transmitted vertically and horizontally in different efficiency
To investigate potential vertical transmission of PfHV1 in P. fici strain AH1-1, individual conidia were isolated from mycelia and cultured on PDA. A total of 51 single subisolates were randomly selected and analyzed for the presence of PfHV1 dsRNAs. The experiments showed

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A special satellite-like RNA all (100%) of the subisolates were infected with PfHV1 dsRNA1 as detected by agarose gel analysis of their nucleic acids after treated by DNase I (Fig 5A). This was further confirmed by dot blotting analysis of 25 randomly selected subisolates using riboprobe 1 and RT-PCR analysis of 8 subisolates using primers PfHV1-1F/1R for dsRNA 1, respectively (Fig 5B). It is worth noting that the RT-PCR products of PfHV1 dsRNA1 showed fewer contents for all the derived RT-PCR detection with primer pair PfHV1-1F/-1R (II, S1 Table) of PfHV1 in some randomly selected conidium-generated subisolates. (C) Electrophoresis analysis on 1% agarose gel of dsRNA-enriched fractions (without treatment by DNase I) and RT-PCR amplicons with primer pair PfHV1-1F/-1R of PfHV1 in the subisolates (AH1-1 hyg V + -1 to -4) of AH1-1 hyg V -(as a recipient strain) contact culture with AH1-1 (donor). Since the fungal genomic bands were kept in the same electrophoresis positions of dsRNA1, the PfHV1 presentence was detected by RT-PCR correspondingly. (Note: D-RNA and dsRNA3 turned faint for unknown reason but they were in the strains identified by both RT-PCR and dot blotting). https://doi.org/10.1371/journal.ppat.1010889.g005

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A special satellite-like RNA subisolates as compared with the parental strain AH1-1 (Fig 5B) whereas dsRNAs 2 or 3 were not detected in all these conidium-generated subisolates, suggesting that they are not vertically transmitted via conidia.
These results suggest that PfHV1 dsRNA1 is efficiently transmitted vertically and horizontally in the host strain, while not easily to other Pestalotiopsis strains. DsRNA2 is efficiently transmitted horizontally but not vertically in the same host strain, whereas dsRNA3 could not be transmitted horizontally and vertically.

PfHV1 confers no obvious effects on the biological traits of the host fungus
To check whether PfHV1 confers some biological effects on the host fungus, the morphologies and growth rates were accessed for strain AH1-1 (PfHV1 + ) and its cured subisolate AH1-1V -(PfHV1 -) after culture on PDA in parallel at 4 dpi, no obvious difference was observed in these aspects (Fig 6A and 6B). Moreover, they induced similar sized lesions as being inoculated on wounded tea leaves (C. sinensis var. Fuyun no.6) ( Fig 6C). As compared with other strains free of PfHV1 including Pestalotiopsis sp. strains (AH1-1-14, TP-2-2W, JWX-3-1, and CJB-4-1), they also showed similar morphologies, growth rates, and virulence (Fig 6D and 6E). These results suggest that PfHV1 confers no obvious effects on the morphologies and virulence of P. fici.

Discussion
In this study, three dsRNA components were detected in P. fici, and their full-length sequences were determined and characterized, suggesting that they belong to a novel hypovirus, tentatively named PfHV1, under the genus Alphahypovirus [3,4,30]. Here, the dsRNA nature of PfHV1 genome was confirmed with enzymatic treatments, suggesting that PfHV1 genomic components accumulate in dsRNA nature in vivo in abundant. The predominant dsRNA form found in infected mycelia appears to be replicative intermediate or replicative form RNA, while the accumulation of plus strand RNA is low due to the RNA silencing antiviral defense response of the fungal host [31]. Other than CHV 1 to CHV4 which have been well studied in Cryphonectria parasitica [1], hypoviruses or hypo-like viruses have been largely discovered from other phytopathogenic fungi, including Sclerotinia sclerotiorum [4,11,30,32], Valsa ceratosperma [3], Fusarium graminearum [8,12], Phomopsis longicolla [33], Macrophomina phaseolina [34], Botrytis cinerea [7], and Rosellinia necatrix [35], and even in non-fungal eukaryotes (invertebrates) [36] revealed by large-scale meta-transcriptomic analysis. To our knowledge, this is the first report of hypovirus isolated from Pestalotiopsis fungus, and the second mycovirus besides of a chrysovirus infecting this fungal genus.
Genomic analysis of PfHV1 dsRNA1 suggests that it contains one ORF coding a polyprotein, which is somewhat different from other members in "Alphahypovirus", since the group genomes typically harbor two ORFs (A and B), with two papain-like cysteine Pro encoded by ORF A and the leading regions of ORF B, respectively, as exemplified by CHV1 [28,37,38], while it is similar to the genomic organization of AaHV1 and those of genera Betahypovirus and Gammahypovirus. Moreover, a complete papain-like cysteine Pro motif was not detected

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A special satellite-like RNA

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in the polyprotein of PfHV1, whereas alignment with the polyproteins of other hypoviruses uncovered the presence of two conserved cysteine Pro core residues (cysteine, and glycine in Fig 3B;) [22] but lacked the histidine at its N-terminal region (29-132 aa region), conserved among hypo-and hypo-like viruses [6], suggesting that the cysteine Pro might be encoded by PfHV1 dsRNA1 to process the polyprotein, but it possess an obvious diversity and evolution at this region of PfHV1 genome. Moreover, the conserved domains of ORFs coding for the functional proteins including RdRp and Hel were detected in the polyprotein of PfHV1. Of these, RdRp is important for RNA viral replication and always conserved in polyproteins encoded by hypo-and hypo-like viruses [6,29]. While several aa were divergent in the conserved motifs as compared with the known members, e.g., in RdRp motif IV and VI. More importantly, a conserved SDD tripeptide was observed in RdRp motif IV, and it is different from most other known (+) ssRNA viruses, in which the consensus tripeptide is GDD [39,40]. dsRNA 2 is determined as a defective component of dsRNA 1, which is very stable since it could be horizontally transmitted, as well as easily observed in other natural Pestalotiopsis spp. strains, like the defective RNA 2 associated with CHV3 [41]. However, dsRNA 2 could not be vertically transmitted together with dsRNA1, suggesting that is not a normal and necessary component of PfHV1 but rather a by-product of virus replication. It is worth noting that it is composed of two inversed sequences of considerately large size, suggesting that a deletionjunction process had happened after the transcription of dsRNA1, in which the defective RNAs is maintenance of an ORF through a deletion-junction to maintain the translation of two different genes [42,43]. The coding capacity of the defective RNA and ability to be translated may affect defective RNA accumulation [44,45], since we have observed the PfHV1 dsRNA1 turned into an obviously lower titer after vertical transmission in the fungal progeny subisolates without the presence of the dsRNA2 components. It is unknown what viral factors are involved in generation and maintenance of the defective dsRNA, which is likely cleaved by dicer-like 2 (DCL2) and argonaute-like 2 (AGL2) proteins responsible for the antiviral RNA silencing pathways in a filamentous fungus [10,46]. dsRNA3 segment is determined to be a SatL-RNA of PfHV1 due to the following reasons: 1) it was co-precipitated with dsRNAs 1 and 2 in the same sucrose fraction of 15% by ultracentrifuge, suggesting that it is encapsulated together with PfHV1 genomic dsRNAs; 2) it codes no structural proteins or RdRp proteins, suggesting its replication depends on the helper virus; 3) no dsRNA3 component was solely observed in any subisolates absent of dsRNA1 after vertical or horizontal transmission. Until now, three satellite or SatL-RNAs have been observed in association with hypoviruses, i.e., dsRNA4 (937 bp in size) and dsRNA3 (dimeric dsRNA4 linking by a poly (A) of CHV3 in C. parasitica and S-dsRNA (3.6 kb) of SsHV1 in S. sclerotiorum, both of which encode authentic proteins and contain a poly (A) tail [11,41]. Of these, CHV3 dsRNA-4 was confirmed to encode a small polypeptide of 9.4 kDa with an in vitro translation experiment [41,47]; S-dsRNA (3.6 kb) of SsHV1 was proposed to encode a protein of 639 aa with an approximate molecular mass of 71 kDa [11]. PfHV1 dsRNA3 shares no detectable identities with the both satellite (or satellite-like) RNAs, contains no a poly (A) tail, and most likely encodes no proteins, suggesting that dsRNA3 is a completely different SatL-RNA as compared the ones of CHV3 and SsHV1, and belongs to a new class of satellite nucleic acids. Generally, satellites are distinct from their helper virus with a nucleotide sequence that is substantially different from that of their helper virus, generally having very little or no sequence similarity with helper viruses, encapsidated by the capsid protein (CP) of helper viruses [48]. Here, dsRNA3 should be encapsulated in the replication vesicles, composed of host-derived lipids [49], instead of CP by the helper virus PfHV1 since the latter has no structural proteins; moreover, dsRNA3 shares a substantially identical sequence (170 bp in size) with the helper virus genome, suggest that it partially originated from its helper virus and

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A special satellite-like RNA is distinct from a typical satellite nucleic acid. Therefore, PfHV1 dsRNA3 represents a novel class of satellite nucleic acids unreported before, unlike the dsRNA satellites in association with Totiviridae and Partitiviridae. We propose these satellite nucleic acids are classified into two classes including those coding dsRNAs with a poly (A) tail (like CHV3 dsRNAs 3 and 4) and those no-coding dsRNAs without a poly (A) tail (like PfHV1 dsRNA3). Additionally, we suggest to broaden the definition of fungal satellites to accommodate these satellite RNAs by including a special type of satellite nucleic acid that has substantial sequence homology with the host viral genome without encapsidation in a coat protein.
Satellite RNAs have been described in association with several mycoviruses in filamentous fungi and yeast [50][51][52], and it has been proved that the amplification used viral RdRp [13,53]. Of them, the satellite RNAs associated with Totiviridae encode a secreted preprotoxin that is lethal to sensitive cells (virus-free or containing helper virus only), and imparts self-protection against the secreted toxin and confers ecological advantage by killing competing virus-or satellite-free fungi; the satellite RNA associated with SsHV1 most likely enhances the hypovirulence traits of S. sclerotiorum. However, the biological functions of other satellite RNAs remain largely unknown. Here, as compared with the dsRNA3-infected and -free strains of P. fici isolates, i.e., strain AH1-1 subisolates derived from horizontal transmission and those from vertical transmission, no obvious morphologies, growth rates or virulence were observed, suggesting that the SatL-RNA has no impact on these aspects. However, as the satellite-like RNA was overexpressed, it inhibits the replication of PfHV1, suggesting that dsRNA3 negatively regulates the replication of PfHV1. These results suggest that the SatL-RNAs harbored by hypoviruses have a similar biological trait to the satellite RNAs associated with animal and plant viruses. E.g., amplification of satellites by viral RdRp may down-regulate synthesis of viral RNAs and expression of their products in the research of Trichomonas vaginalis virus [53]; cucumber mosaic virus (CMV) satellite RNA reduced the yield of accumulated helper virus [54,55]. Based on the in vitro competition assay, the reduction was proposed to be due to the competition of satellite RNA for limited amount of viral replicase with CMV gRNAs [56]. Furthermore, a three-way branched secondary structure was identified in the satellite RNA, and was indispensable for the helper virus inhibition [57]. Similarly, a conserved apical hairpin stem-loop structure was identified in the 5 0 -UTR of bamboo mosaic virus (BaMV) satellite RNA as a potent determinant of the down-regulation of helper virus replication [58]. Since the SatL-RNA encodes no functional proteins, we conclude that it inhibits the replication of helper virus most likely with similar mechanism to the CMV and BaMV satellite RNAs.
In summary, to gain insight into mycoviruses in P. fici, belonging to an important fungus genus related to agriculture, industry and medicine, a novel hypovirus from P. fici together with its D-RNA and a SatL-RNA, were identified and characterized. To our knowledge, PfHV1 represents the first hypovirus and the second mycovirus isolated from Pestalotiopsis spp., and its SatL-RNA represents a novel class of satellite nucleic acids, which should contribute to our better understanding of the taxonomy, evolution, molecular and biological traits of mycoviruses, as well as the related satellites.
Supporting information S1

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A special satellite-like RNA