Characterization of a novel double-stranded RNA mycovirus conferring hypovirulence from the phytopathogenic fungus Botryosphaeria dothidea

Virology 493 (2016) 75–85

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Characterization of a novel double-stranded RNA mycovirus conferring hypovirulence from the phytopathogenic fungus Botryosphaeria dothidea Lifeng Zhai a,b, Jun Xiang b, Meixin Zhang b, Min Fu b, Zuokun Yang b, Ni Hong a,b,c, Guoping Wang a,b,c,n a b c

National Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University, Wuhan 430070, China

art ic l e i nf o

a b s t r a c t

Article history: Received 19 December 2015 Returned to author for revisions 26 February 2016 Accepted 14 March 2016 Available online 22 March 2016

A novel double-stranded RNA (dsRNA) virus, designated as Botryosphaeria dothidea RNA virus 1 (BdRV1), isolated from a hypovirulent strain YZN115 of Botryosphaeria dothidea was biologically and molecularly characterized. The genome of BdRV1 comprises of five dsRNAs. Each dsRNA contains a single open reading frame. The proteins encoded by dsRNA1-4 shared significant amino acid identities of 55%, 47%, 43% and 53% with the corresponding proteins of Aspergillus fumigatus tetramycovirus-1. DsRNA1, 3, and 4 of BdRV1 encoded an RNA-dependent RNA polymerase, a viral methyltransferase, and a P-A-S-rich protein, respectively. Function of proteins encoded by the dsRNA2 and dsRNA5 were unknown. BdRV1 conferred hypovirulence for its host and could be transmitted through conidia and hyphae contact. & 2016 Elsevier Inc. All rights reserved.

Keywords: Botryosphaeria dothidea Hypovirulence Double-stranded RNA virus Genome

1. Introduction Mycoviruses have been found in all major fungal groups (Ghabrial and Suzuki, 2009; Pearson et al., 2009; Xie and Jiang, 2014). Ever since Cryphonectria parasitica hypovirus 1 (CHV1), a hypovirulence-associated mycovirus that infects the fungus Cryphonectria parasitica, was successfully used to control chestnut blight in Europe (Anagnostakis, 1982; MacDonald and Fulbdght, 1991; Nuss, 1992; Shapira et al., 1991), increasing mycoviruses have been reported (Ghabrial and Suzuki, 2009; Xie and Jiang, 2014). Most mycoviruses have double-stranded (ds) or positivesense single-stranded (þss) RNA genomes. However, the genomes of some mycoviruses consist of DNA (Yu et al., 2010) or negativesense RNA (  ssRNA) (Liu et al., 2014). The currently known dsRNA mycoviruses were classified into six families, including Chrysoviridae, Megabirnaviridae, Partitiviridae, Quadriviridae, Reoviridae, and Totiviridae (Ghabrial et al., 2015). The genetic materials of most dsRNA mycoviruses are packaged in isometric particles (Ghabrial et al., 2015; Ghabrial and Suzuki, 2009), and some dsRNA viruses lack a conventional virion (Cai et al., 2013; Spear et al., 2010). n Corresponding author at: National Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, China. E-mail address: [email protected] (G. Wang).

http://dx.doi.org/10.1016/j.virol.2016.03.012 0042-6822/& 2016 Elsevier Inc. All rights reserved.

Recently, a dsRNA virus, Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) infecting the human pathogenic fungus Aspergillus fumigatus, has been reported. The virus is a unique ribonucleoprotein form, in which protein encoded by the virus apparently coated but did not encapsidate the viral genome (Kanhayuwa et al., 2015). The dsRNA1 of AfuTmV-1 encodes an RNA-dependent RNA polymerase (RdRp), which contains a unique GDNQ motif as a normal characteristic in some mononegavirales with nonsegmented  ssRNA genomes. The dsRNA3 encodes an S-adenosyl methionine-dependent methyltransferase capping enzyme and the dsRNA4 encodes a proline–alanine–serine rich protein (PASrp) that apparently coats but does not encapsidate the viral genome (Kanhayuwa et al., 2015). In the most cases, mycoviruses cause mild or no obvious effects on the growth of their fungal hosts, but some greatly reduce virulence and suppress the growth of their hosts (Anagnostakis, 1982; Ghabrial and Suzuki, 2009; MacDonald and Fulbdght, 1991; Pearson et al., 2009; Wang et al., 2014; Wu et al., 2012; Xiao et al., 2014; Xie and Jiang, 2014). The fact that hypovirulence-associated mycoviruses reduce the severity of fungal diseases has attracted the attentions of many phytopathologists (Ghabrial and Suzuki, 2009; Nuss, 1992; Pearson et al., 2009; Xie and Jiang, 2014; Yu et al., 2013). In one classical instance of mycovirus-based biocontrol, CHV1 was successfully implemented in Europe to reduce the

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impact of chestnut blight disease (Van Alfen et al., 1975). Recently, Rosellinia necatrix megabirnavirus 1 (RnMBV1) have shown an exploitable potential for controlling white root rot of fruit trees (Chiba et al., 2009). Sclerotinia sclerotiorum partitivirus 1 (SsPV1/ WF-1) and Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) could be exploited as a potential biocontrol agents of diseases caused by Sclerotinia sclerotiorum (Xiao et al., 2014; Yu et al., 2013). Botryosphaeria dothidea infects a large number of economically important fruit and wood trees and causes fruit rots, stem warts, stem canker and twig dieback (Slippers and Wingfield, 2007; Tang et al., 2012; Yan et al., 2013; Zhai et al., 2014). Pear trees are perennial plants and most cultivars are susceptible to B. dothidea. Recently, three dsRNA mycoviruses infecting B. dothidea strains were sequenced (Wang et al., 2014; Zhai et al., 2015). Among these viruses, Botryosphaeria dothidea chrysovirus 1 (BdCV1), a chrysovirus, might confer the hypovirulence to B. dothidea strain LW-1 (Wang et al., 2014). In this study, we characterized a novel dsRNA mycovirus isolated from a strain YZN115 of B. dothidea. The virus was named as Botryosphaeria dothidea RNA virus 1 (BdRV1). BdRV1 had a similar genome structure and close phylogenetic relationships with AfuTmV-1. Importantly, BdRV1 conferred hypovirulence to the host B. dothidea and could be transmitted via asexual sporulation at 100% rate. These characteristics permit to use the virus as a potential biological control agent for the diseases caused by B. dothidea.

2. Materials and methods 2.1. Fungal isolates and culture conditions B. dothidea strain YZN115, a virus source, was isolated from a wart-diseased stem tissue of pear (Pyrus bretschneideri cv. ‘Suli’) grown in Henan province in China. B. dothidea strains JNT1111 and EJ23011, which were isolated from Shanxi and Huibei province in China, respectively, and they had high virulence on pear fruit and shoot and different colonial morphology (Zhai et al., 2014), were used as controls in assessing biological features of strain YZN115 in this study. The other 187 strains of B. dothidea, 42 strains of B. parva, 11 strains of B. obtusa and 3 strians of B. rhonida isolated from pear plants grown at 18 provinces in China (Zhai et al., 2014), were used for assessment of the presence of the virus. All those strains were cultured on PDA at 25 °C in the dark. Mycelial agar discs (5 mm) were stored in a sterilized 25% glycerol solution at 80 °C. 2.2. The extraction and purification of dsRNA All tested fungal strains were cultured on cellophane membranes overlaid on the surfaces of PDA plates for 4 days. The mycelia were harvested and ground to a fine powder in liquid nitrogen. DsRNA was extracted using a patented method of our lab (No. ZL201310072994.3). All dsRNA preparations were digested with DNase I and S1 nuclease (Takara, Dalian, China) to eliminate DNA and single-stranded RNA (ssRNA) contaminants and subjected to electrophoresis in a 1.5% (w/v) agarose gel at 75 V. The gel was stained with 0.1 μg/mL ethidium bromide and viewed in an UV trans-illuminator. The separated dsRNAs were excised from the gel and purified using a DNA Gel Extraction Kit (Axygen Scientific, Inc. Wujiang, China). Purified dsRNAs were dissolved in diethypyrocarbonate (DEPC)-treated water and kept at  80 °C.

2.3. cDNA synthesis and molecular cloning The purified dsRNAs were used as templates to synthesize the first-strand cDNAs using the moloney murine leukemia virus (MMLV) reverse transcriptase according to the manufacturer's instructions (Fermentas, USA). The second-strand cDNAs were synthesized as described by Sambrook et al. (Herrero et al., 2011), purified by using chloroform and isoamyl alcohol, and A-tailed by co-incubated with Taq DNA polymerase and dNTPs (TaKaRa, Dalian, China). The resulting double stranded cDNAs were ligated into the pMD18-T vector (TaKaRa, Dalian, China) and transformed into competent cells of Escherichia coli DH5α. All positive clones with inserts more than 500 bp were selected and sequenced. Sequence gaps between clones were determined by RT-PCR using primers designed on obtained cDNA sequences. For the determination of the terminal sequences of each dsRNAs, the 30 -terminus of each strand of dsRNAs was ligated with the closed adaptor primer RACE-OLIGO [50 -(P)-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2) 30 ] using T4 RNA ligase (TaKaRa, Dalian, Ltd, China) at 8 °C for 18 h. The oligonucleotide-ligated dsRNA was reverse transcribed with MMLV reverse transcriptase and 10 pmol of a primer complementary to the oligonucleotide RACE-OLIGO primer (oligo RACE-1st, 50 -GGCAATTAACCCTCACTAAAG-30 ). The cDNA was amplified using another primer complementary to the RNA ligation oligonucleotide (ORACE-2nd:50 -TCACTAAA0 GAATTCGATCGATC-3 ) and the sequence-specific primers corresponding to the 50 - and 30 -terminal sequences of the dsRNAs and LA Taq DNA polymerase. The amplified products were cloned. Sequencing was performed at Genscript Biotechnology Co., Ltd., Nanjing, China. At least three independent clones of each product were determined in both orientations. 2.4. Genome organization and sequence analysis Potential open reading frames (ORFs) were identified using the DNAMAN software package (DNAMAN version 6.0; Lynnon Biosoft, Montreal, Canada). The stem-loop structures of terminal sequences of the viral RNAs were predicted using the program RNA folding with the default settings at the Mfold website (http:// mfold.rna.albany.edu/?q ¼mfold/RNA-Folding-Form2. 3) (Zuker, 2003). Conserved domains of full-length cDNA sequence of the virus were identified using CD-search on the website of the National Center for Biotechnology Information (NCBI) (http:// www.ncbi.nlm.nih.gov/) and the motif scan website (http://www. genome.jp/tools/motif/). Multiple alignments of nucleotide and amino acid sequences were conducted by using the MAFFT version 7 (Katoh and Standley, 2013). The results were visualized using BoxShade program (http://www.ch.embnet.org/software/BOX_ form.html). The best-fit model of protein evolution (LG þG) was determined by maximum likelihood analyses using Molecular Evolutionary Genetics Analysis Computational Core (MEGA6-CC) (Kumar et al., 2012). Phylogenetic trees were generated using the maximum-likelihood method basing on these alignments and the best-fit model, as computed with MEGA6-CC. Reference sequences of viruses used for comparative analyses were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/genomes) (Table 1). 2.5. Biological tests Freshly grown mycelial agar plugs were transferred onto PDA medium, and cultured at 25 °C for 10 days. The colony morphology was observed and the diameters of mycelial colonies were measured every day using a crisscross method until the plates were filled with colony. Each of the above tests has three replicates. The pathogenicity of each strain was determined by mycelial

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Table 1 The sources and GenBank accession numbers of viruses used for sequence alignment and phylogenetic analysis of their RdRps. Viruses

Abbreviation

Host

Genus

Family

GenBank accession no.

Aspergillus fumigatus tetramycovirus-1 Cladosporium cladosporioides virus 1 Alternaria tenuissima virus Helminthosporium victoriae virus 190 S Penicillium chrysogenum virus Cryphonectria hypovirus 1 Cryphonectria hypovirus 2-NB58 Cryphonectria hypovirus 3 Cryphonectria hypovirus 4 Phomopsis longicolla hypovirus Sclerotinia sclerotiorum hypovirus 1 Valsa ceratosperma hypovirus 1 Duck astrovirus C-NGB Avastrovirus 1 Mamastrovirus 1 Mamastrovirus 13 Beet cryptic virus 1 Vicia cryptic virus Rosellinia necatrix partitivirus 2 Rosellinia necatrix partitivirus 4 Heterobasidion partitivirus 8 Aspergillus fumigatus partitivirus 1 Ustilaginoidea virens partitivirus 1 Ustilaginoidea virens partitivirus 2 Pepper cryptic virus 1 Pepper cryptic virus 2 Raphanus sativus cryptic virus 3

AfuTmV-1 CcRV1 AtRV HvV190S PcV CHV1 CHV2-NB58 CHV3 CHV4 PlHV1 SsHV1 VcHV1 DAstV-1 TAstV HAstV OAstV BCV1 VCV RnPV2 RnPV4 HetRV8 AfuPV1 UvPV1 UvPV2 PepCV1 PepCV2 RsCV3

Fungus Fungus Fungus Fungus Fungus Fungus Fungus Fungus Fungus Fungus Fungus Fungus Animal Animal Human Animal Plant Plant Fungus Fungus Fungus Fungus Fungus Fungus Plant Plant Plant

Unassigned Unassigned Unassigned Victorivirus Chrysovirus Hypovirus Hypovirus Hypovirus Hypovirus Hypovirus Hypovirus Hypovirus Avastrovirus Avastrovirus Mamastrovirus Mamastrovirus Alphapartitivirus Alphapartitivirus Alphapartitivirus Betapartitivirus Betapartitivirus Gammapartitivirus Gammapartitivirus Gammapartitivirus Deltapartitivirus Deltapartitivirus Deltapartitivirus

Unassigned Unassigned Unassigned Totiviridae Chrysoviridae Hypoviridae Hypoviridae Hypoviridae Hypoviridae Hypoviridae Hypoviridae Hypoviridae Astroviridae Astroviridae Astroviridae Astroviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae Partitiviridae

CDP74618 AII80567 AJP08049 NP_619670 AF296439 AAA67458 AAA20137 AF188515 AY307099 AIG94930 AEL99352 BAM08994 YP_002728002 CAB95006 NP_751905 CAB95003 EU489061 AY751737 AB569997 AB698493 AFW17810 FN376847 KC503898 KF361014 JN117276 JN117278 FJ461349

inoculation on detached fruits and shoots. The matured fruits of pear (P. bretschneideri cv. Huangguan) and apple (Malus domestica cv. Fuji) and one-year-old shoots from pear (Pyrus pyrifolia cv. Hohsui, P. pyrifolia cv. Nijisseiki, and P. communis cv. Lecounte) and apple (M. domestica cv. Fuji) trees were inoculated using colonized agar plugs (5 mm in diameter) as described previously excepting that the wounds were 5 mm in diameter on shoots (Zhai et al., 2014). The diameters of the fruit lesions were recorded daily up to five days post inoculation (dpi). The lengths of canker lesions on shoots were recorded at 12 dpi. Each of the above tests has three replicates. 2.6. Vertical transmission of hypovirulence-associated dsRNAs Conidia of YZN115 were induced on PDA plate under alternating cycles of light/dark (16 h:8 h) and fluorescent light as a previously described (Zhai et al., 2014). Conidia was individually picked out and transferred into fresh PDA plates. The presence of the dsRNAs in mycelia of single-conidian strains was assessed. 2.7. Horizontal transmission of hypovirulence-associated dsRNAs Horizontal transmission assay was executed on PDA in petri dishes using the hyphal contact technique as described previously (Zhang and Nuss, 2008). The strain YZN115 served as the donor, and 88 strains which lacked detectable dsRNAs served as recipients. The dual cultures were incubated for 10 days at 25 °C. Mycelial derivative isolates were obtained by re-culturing the mycelial agar plugs from the margin of recipients (the farthest site from strain YZN115 colony). The presence of dsRNA segments in each mycelial derivative isolate was determined. Furthermore, three JNT1111 derivatives (Y15-J111-1, Y15-J111-2, and Y15-J111-3) were individually purified by single hyphal tip culturing and used to assess the effects of virus infection on its biological traits. Colony morphology, growth rates, and virulence of the derivatives were evaluated as described above and compared with those of their parental strains YZN1115 and JNT1111. To exclude for the co-

presence of parental YZN115 in the three sub-strains, the universally primed polymerase chain reaction (UP-PCR) analysis was implemented using a single primer AS15inv (50 -CATTGCTGGCGAATCGG-30 ), as described previously (Naumova et al., 2003). 2.8. Virus purification and peptide mass fingerprinting (PMF) analysis of viral proteins The viral like particles (VLPs) was purified from strain YZN115 as described by Tang et al. (Tang et al., 2010). Fungal strain YZN115 was for 4–5 days. Approximately 30 g mycelia cultured on cellophane membranes overlaid on PDA for 4–5 days were harvested and ground to a fine powder in liquid nitrogen. The powder was transformed into a conical flask containing 120 ml of phosphate buffer (PB, 0.1 M, pH 7.4 containing 0.1% β-mercaptoethanol) and shaked at 150 rpm for 30 min at 4 °C and centrifuged at 5000 g for 20 min. The supernatant was centrifuged at 10,000  g for 20 min to remove the hyphal cell debris. This step was repeated once. The final supernatant was centrifuged at 100,000  g for 1.5 h (Optima LE-80K, Backman Coulter, Inc. U.S.). The resulted pellet was resuspended in 0.5 ml of PB buffer. After centrifugation at 10,000  g for 1 min, the supernatant was overlaid on 10 to 40% (wt/vol) sucrose gradient and centrifuged at 70,000  g at 4 °C for 3 h. Aliquots about 1 mL were individually collected and total RNAs were isolated from each fraction. The presence of the viral dsRNAs in the preparations was extracted with chloroform and isoamyl alcohol and determined by agarose gel electrophoresis. The fractions containing viral dsRNAs were centrifuged at 100,000  g for 2 h and precipitate was suspended in 50 μl of 0.05 M PB buffer (pH 7.4). The supernatants were visualized under a transmission electron microscope (TEM, H7650; Hitachi). The proteins from final suspension were detected by electrophoresis on a 12% (wt/vol) polyacrylamide gel amended with 1% (wt/vol) sodium dodecyl sulfate (SDS). After stain with Coomassie brilliant blue R-250 (Bio-Safe CBB; Bio-Rad, USA), the gel stripe with protein bands were excised subjected to peptide mass fingerprinting (PMF) analysis at Sangon Biotech (Shanghai, China).

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2.9. Expression of hexahistidine-tagged ORF4 protein in E. coli and polyclonal antibodies production

3. Results 3.1. Biological characterization of B. dothidea strain YZN115

The total RNA extracted from the virus preparation was used as template for the RT-PCR amplification ORF4 of BdRV1/YZN115 by using a specific primer pair RNA4-BanHI-F (50 -CGCGGATCCATGGCCTCAACCACCACCACCAAC-30 ) and RNA4-HindIII-R (50 CCCAAGCTTTCACTCGGACGAAGGACCTGCAGT-30 ). The PCR product was purified and ligated into the vector pET-28a( þ). The cells of E. coli strain Rosetta (DE3) transformed with recombinant plasmid were cultured in LB under the induction of 1 mM Isopropyl, 1-thioβ-D-galactopyranoside (IPTG) at a speed of 220 rpm at 16 °C for 12–16 h. Recombinant proteins were purified using Ni-NTA agarose (Qiagen, Hilden, Germany). Briefly, bacterial cells were suspended in Lysis Buffer (50 mM Na2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0), disrupted by ultrasound (200 W, each for 2 s with 2 s intervals, for 30 min in total) on ice and then centrifuged at 10,000  g for 20 min at 4 °C. The supernatant containing fusion protein was then passed through a pre-equilibrated column. After washing, the fusion protein bound on the column were eluted and desalted against phosphate-buffered saline. The purified recombinant protein was detected by 12% SDS-PAGE. The polyclonal antibodies (PAb-p4) were generated by three times of injections of the recombinant protein into New Zealand rabbits by Genscript Biotechnology Co., Ltd. (Nanjing, China). 2.10. Western blotting and indirect ELISA Total proteins were extracted from strains YZN115, Y15-J111-1, and JNT1111 as reported previously (Wang et al., 2006). The proteins separated in 12% SDS-PAGE were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Then, the membrane was blocked with 5% (w/v) skim milk in TBST buffer overnight at 4 °C and incubated in the prepared PAb-p4 at a dilution of 1:10,000, and followed by incubation with anti-rabbit IgG (1:10,000 dilution) conjugated to alkaline phosphatase (AP). Finally, BCIP-NBT (5-bromo-4-chloro-3-indolylphos-phate ptoluidine salt-Nitro Blue Tetrazolium chloride) was added as the substrate for color development. Indirect enzyme linked immunosorbent assays (ELISA) was carried out to detect the P4 of BdRV1/YZN115 in the extracts of mycelia YZN115 by using the prepared PAb-p4 as previously described methods (Wu et al., 2014). 2.11. Immuunosorbent electron microscopy analysis Purified virus preparations were tested by immuuno-capture electron microscopy (ISEM) analysis (Agranovsky et al., 1995). The carbon-coated grids were incubated with BdRV1 PAb-p4 (at a dilution of 1:500), rinsed with drops of 0.1 M PB, and then placed on a droplet of viral preparation. After rinsing and incubation with PAb-p4 (at dilution 1:100), the grids were incubated with 10 nm gold particles conjugated goat anti-rabbit IgG, stained with 2% phosphotungstic acid and visualized under TEM. 2.12. Statistical analysis The data were analyzed using SAS software (version 9.0; SAS Institute). The variances between pairs of treatments were calculated using the Fisher's least significant difference test at significance level of P o0.05.

On PDA medium, B. dothidea strain YZN115 showed a much slow growth speed as compared with the virus-free B. dothidea strains JNT1111 and EJ23011 and its colony consisted of thin aerial hypha with sectored regions at the edge of the medium (Fig. 1A). When the colonized plugs of the three strains were inoculated onto fruits of pear and apple, the strain YZN115 induced soft rotting lesions on fruits with much less diameters than that induced by virus-free strains JNT1111 and EJ23011 (Fig. 1B). Accordingly, on detached shoot, the necrotic cankers induced by strain YZN115 were less than 4 mm in lengths and the incidence rate was less than 10%, and virus-free stains JNT1111and EJ23011 induced black cankers of 5–27 mm and 5–25 mm in length, respectively and showed 100% incidence at 12 dpi. The results indicated that the B. dothidea strain YZN115 was hypovirulent in fruits and shoots of pear and apple. 3.2. Genetic organization of dsRNAs in B. dothidea strain YZN115 Five distinct dsRNA segments with estimating sizes of 1.2– 3.0 kb were detected in strain YZN115, and these dsRNAs were not detected in strain JNT1111 (Fig. 2A). These segments were recovered from the agarose gel and subjected to cDNA cloning and sequence analysis. The complete nucleotide sequences of the five dsRNA components were obtained by sequencing the ds-cDNAs library, gapfilling RT-PCR clones, and RACE clones. A schematic representation of the genetic organization of the coding strands of dsRNAs1–5 is shown in Fig. 2B. Sequence analysis for the full-length cDNAs of dsRNAs1–5 revealed that the entire lengths of each dsRNA were 2397, 2184, 1967, 1131 and 1060 bp, and each contained a single ORF. Correspondingly, ORF1 to ORF5 putatively encoded proteins (P1–P5) of 757, 649, 572, 257 and 279 amino acids (aa) with calculated molecular masses of 82, 75, 62, 28, and 30 kDa, respectively. The proteins P1–P4 shared significant aa identities of 55%, 47%, 43%, 53%, with corresponding proteins of AfuTmV-1 (Kanhayuwa et al., 2015). However, the P5 has no similarities to any known proteins. The 50 -untranslated regions (UTRs) of the coding strands of dsRNAs 1–5 were 53, 71, 57, 89, and 98 nt in length. The 30 -UTRs were 70, 29, 191, 214, and 132 nt in length, respectively. The 50 and 30 - termini of coding strands of the five dsRNAs contained conserved 9 nt sequence (CGAUUAAAA) and 6 nt sequence (UGGGGU), respectively (Fig. 3A). Interestingly, plus-strands of the genomic dsRNAs of this virus and AfuTmV-1 also shared highly similar the 50 - and 30 -terminal sequences, including CGAWUAAAA at the 50 -terminus and YGGGGK at the 30 -terminus, GCUUG at positions 22–26 nt, and high GC content at the 30 -terminal regions (Fig. 3A). As in that of AfuTmV-1, sequences of the 50 - and 30 terminal regions of the virus coding strands were predicted to fold into stable stem-loop structures, as illustrated for dsRNA1 (Fig. 3B). Collectively, these features supported that dsRNAs1–5 represented the genome of a novel mycovirus species, and the virus was assigned as Botryosphaeria dothidea RNA virus 1 (BdRV1). The corresponding sequences of dsRNAs1–5 of BdRV1/YZN115 have been deposited in GenBank under the accession numbers KP245734–KP245738. A homology analysis revealed that the P1 of BdRV1 was most closely related to RdRp encoded by AfuTmV-1 by sharing 55% amino acid sequence identity (Accession no. CDP74618 and E value ¼0.00), and also similar to the putative RdRp of Cladosporium cladosporioides virus 1 (CcRV1, accession no. AII80567, E value ¼0.00 and 45% identity) and Alternaria tenuissima virus

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Fig. 1. Phenotypes on PDA and virulence on fruits of pear and apple of B. dothidea strains. (A) Colony morphology of strains YZN115, JNT1111 and EJ23011 on PDA. (B) Hyphal growth rates of strains YZN115, JNT1111, and EJ23011 on PDA at 25 °C and the average diameters of rot lesion caused by the three strains (25 °C; 5 days post infection) on the fruits of pear (Pyrus bretschneideri cv. Huangguan) and apple (Malus domestica cv. Fuji).

(AtRV, Accession no. AJP08049, E value ¼2e  123 and 47% identity), had low aa sequence identity (26–33%) with polyproteins or RdRps of animal, fungus or plant viruses in the families Astroviridae, Partitiviridae, and Picornaviridae (Supplemental Table S1). Multiple aa alignment of the predicted RdRp showed the presence of several conserved sequence regions in the AfuTmV-1, AtRV, CcRV1 and BdRV1 (Supplemental Fig. S1A). Importantly, the P1 of BdRV1 had five motifs (3–7) conserved in RdRp of dsRNA and ssRNA viruses of simple eukaryotes. However, the GDN triad in the motif 6 was different from SDD in viruses in the family Hypoviridae and GDD in viruses in the families Totiviridae, Chrysoviridea, Partitiviridea, and Astroviridae (Supplemental Fig. S1B). The GDN tetrad followed immediately by a Q residue in RdRP was a sequence characteristic normally found in the proteins encoded by L genes of mononegavirales (Liu et al., 2014; Poch et al., 1990). Phylogenetic analysis basing on the conserved aa sequences of the putative RdRp of BdRV1 and other recognized viruses revealed that BdRV1, AfuTmV-1, CcRV1, and AtRV formed a separated clade from dsRNA viruses. In the clade, BdRV1 was alone from AfuTmV-1, CcRV1, and AtRV (Fig. 4).

The deduced aa sequence of protein P2 of BdRV1 shared the highest identity of 47% with a unknown function protein encoded by dsRNA2 of AfuTmV-1 (accession no. CDP74619 and E value ¼0.0) and the two proteins had identical aa residues (Fig. S3A). In addition, the P2 had low similarity with proteins encoded by a dsRNA virus isolated from Alternaria sp. (accession no. ACL80752, E value ¼1e  163 and 42% identity) and a dsRNA mycovirus isolated from Cladosporium cladosporioides (Accession no. AII80568, E value ¼2e  80 and 32% identity). A BLASTp search for the aa sequences showed that BdRV1 P3 shared the highest identity of 43% with a Methyl transferase encoded by dsRNA3 of AfuTmV-1 (Accession no. CDP74620, and E value ¼6e  131; Supplemental Fig. S3B), and 37% and 25% identities with an unassigned proteins encoded by dsRNA from Melampsora lini (Accession no.CAA45724; E value ¼ 7e  95) and CcRV1 (Accession no. AII80569; E value ¼ 2e  28), respectively. A conserved protease domain of S-adenosylmethionine-dependent methyltransferases (cd02440 and E value ¼1.28e  05) was excavated out in the N-terminal region (positions 129–240) of the P3. The BdRV1 P4 had 53% aa sequence identity with a PASrp encoded by dsRNA4 of AfuTmV-1 (accession no. CDP74621; E

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Fig. 2. DsRNAs extracted from strain YZN115 and genomic organization of Botryosphaeria dothidea RNA 1 (BdRV1). (A) 1.5% agarose gel electrophoretic profiles of dsRNA preparations extracted from YZN115, JNT1111, and EJ23011 after digestion with DNase I and S1 nuclease. (B) Genomic organization of dsRNAs 1–5 of BdRV1.

value ¼ 7e  71; Supplemental Fig. S3C), and 37% identity to a hypothetical protein of CcRV1 (Accession no. AII80570; E value ¼ 3e  31). The P4 contained 7.6% proline (P), 13.5% alanine (A), and 6.2% serine (S) residues out of total amino acid number (257 aa). The PASrp of AfuTmV-1 as colloidal proteinaceous components enveloped infectious dsRNAs (Kanhayuwa et al., 2015). Meanwhile, a conserved motif (aa 203–218) associated with a bipartite nuclear localization signal profile was also presented in the P4 (E value ¼2.1e  4). 3.3. Recovery of dsRNAs and a putative structural protein from the purified virus preparations Agarose gel analysis revealed that the total RNAs extracted from the purified preparations of YZN115 had similar profiles to the dsRNAs extracted from mycelia of strain YZN115 (Supplemental Fig. S2A). Moreover, the insusceptibility to DNase I and resistance to S1 nuclease confirmed that the dsRNA nature of the extracted total RNAs (Supplemental Fig. S2A lane3). SDS-PAGE analysis of purified BdRV1 preparations showed a single major protein band with a molecular mass of  35 kDa (Supplemental Fig. S2B). PMF analysis of the 35 kDa protein generated 13 peptide fragments (Supplemental Table S2), which matched the partial sequence at aa 17–265 of P4 encoded by dsRNA4, accounting for 69% of the entire coverage (257 aa). The results suggested that 35 kDa P4 of BdRV1 might sever as a tentative structural protein. 3.4. The recognition of BdRV1 protein by the polyclonal antibodies against P4 SDS-PAGE analysis revealed that the fusion P4 with an estimated molecular weight of 36.0 kDa was expressed in E. coli cells under the IPTG induction, and was absent in E. coli cells without IPTG induction and transformed with the empty vector pET-28a

(þ). There was only a single protein band in the purified protein preparation (data not show). Western blotting and ELISA analyses using PAb against the P4 from prokaryotic expression system showed that the PAb-p4 recognized strongly a protein about 35 kDa in viral preparation of YZN115, and in protein extracts from mycelium of YZN115 and Y15-J111-1 which obtained from strain JNT1111 in horizontal transmission assays through hyphae contact, but the reaction signal was absent in extracts from BdRV1-free strain JNT1111 (Fig. 5A and B). The result indicated that the PAb-p4 could specifically recognize the native protein of BdRV1. The P4 could be obtained from viral preparation and mycelium of BdRV1-infected strains. The reaction signal could be detected in sub-strain Y15J111-1 revealed that the P4 was a necessary protein in activity of BdRV1. To determine whether the virus particles really exist in the virus preparations, the preparations were subjected to direct TEM and immune-decoration TEM analyses. Bacilliform virus-like particles about 30–80 nm in length and 13.4 7 1.9 nm in diameter, were observed under TEM, but could not be decorated by the PAbp4 (results were not shown), indicating that the P4 might not encapsidate the dsRNAs of BdRV1/YZN115 as virions, a similar phenomenon happened for AfuTmV-1 (Kanhayuwa et al., 2015). 3.5. Transmissibility of BdRV1 and its association with hypovirulence Totally, five hundreds of single-conidium strains were derived from YZN115. Analyzes of dsRNAs revealed that five dsRNA components of BdRV1 were recovered from all these sub-strains. Twelve sub-strains selected randomly were subjected to bioassays. Surprisingly, the growth rates on PDA of these sub-strains were significant faster than the growth rate of strain YZN115 (26.8– 30.2 mm/d vs 20.8 mm/d) (Supplemental Table S3). However, the virulence of these sub-strains on pear fruits (P. bretschneideri cv.

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Fig. 3. Multiple sequence alignments and the predicted secondary structures for the terminal regions of the coding strand of BdRV1. (A) Conserved sequences of the 50 termini and 30 -termini of the dsRNAs of BdRV1 and AfuTmV-1. Black, grey, and light grey backgrounds indicate denote nucleotide identity no less than 100%, 80%, and 60%, respectively. (B) The secondary structures proposed for dsRNA1 of BdRV1 with the lowest energy (http://mfold.rna.albany.edu/?q ¼ mfold/RNA-Folding-Form2.3).

Huangguan) and their mother strain YZN115 did not have significant difference (Supplemental Table S3). In horizontal transmission assays, 88 dsRNA-free B. dothidea strains were used as recipients in the hyphal contact with the strain YZN115. The five dsRNA elements of BdRV1 were recovered in derivatives from nine recipient strains. The presence of the viral dsRNAs in these derivatives was confirmed by RT-PCR using the primers specific for the BdRV1 dsRNA1 and sequencing of amplification products. As representatives, the derivatives from recipient strain JNT1111 were identified by lacking a band, which presented in YZN115 in UP-PCR analysis (Supplemental Fig. S4). On PDA, as compared to their mother strain JNT1111, derivatives Y15J111-1, Y15-J111-2, and Y15-J111-3 infected by BdRV1 showed slow growth speed and their colony margins were sectored (Fig. 6A; Table 2), and even did not produce any pycnidium after incubation for 60 days. These results revealed that the impaired growth rates were correlated with transmission of BdRV1. In accordance, substrains Y15-J111-1, Y15-J111-2, and Y15-J111-3 did not cause disease lesions, but strain JNT1111 caused large black cankers on the shoots of pear and apple (Fig. 6B; Table 2). Similarly, these strains did not cause lesion (Y15-J111-1and Y15-J111-3) or caused small rot lesion (Y15-J111-2, average 11.1 mm) on pear and apple fruits at

5 dpi (Fig. 6C; Table 2). While, their mother strain JNT1111 caused severe fruit rot, which reached the cores of pear and apple fruits at 5 dpi (Fig. 6C). Together, BdRV1/YZN115 in strain YZN115 can be transmitted vertically from mother mycelia to conidial generations and transmitted horizontally to other strains through hyphae contact. The transmissions of virus BdRV1 also confer hypervirulence to substrains efficiently. 3.6. Occurrence and sequence polymorphisms of BdRV1 in B. dothidea strains To examine the natural occurrence and geographical distribution of BdRV1 in four Botryosphaeria species isolated from pear and apple trees in China, dsRNA was extracted from hyphae cultures of 187 strains of B. dothidea, 42 strains of B. parva, 11 strains of B. obtusa and 3 strians of B. rhonida. Electrophoresis analysis revealed the presence of dsRNAs in 95 B. dothidea strains, but not in the strains of other three species. Of the 95 B. dothidea strains, 13 strains contained five dsRNA elements with sizes 1.2–3.0 kbp, similar to the genomic dsRNAs BdRV1/YZN115 (Supplemental Fig. S5, upper panel). Furthermore, RT-PCR using the primer specific

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4. Discussion

Fig. 4. Maximum-likelihood phylogenetic tree constructed basing on the complete amino acid sequences of the RdRp of BdRV1 and other selected members of families Astroviridae, Chrysoviridae, Hypoviridae, Partitiviridae, and Totiviridae. The best-fit model of protein evolution (LG þ G) was used for computing the tree. Bootstrap values (relative) obtained with 1000 replicates are indicated on the branches, and branch lengths and the scale bar on the bottom left both correspond to genetic distance. Abbreviations of the virus names o and GenBank accession numbers are listed in Table 1.

for dsRNA1 of BdRV1/YZN115 confirmed the BdRV1 infection in these strains (Supplemental Fig. S5, low panel). Amplicons of 560 bp from these strains shared greater than 87% nt identity with the corresponding fragment of BdRV1 (data not show). The BdRV1 dsRNAs alone- or co-existed with other dsRNA elements in B. dothidea strains (Supplemental Fig. S5). The co-existences of different dsRNA elements suggested that BdRV1 could co-infect B. dothidea with other viruses. The growth rates of the 13 strains varied from 2.8 mm/d to 42.5 mm/d on PDA medium at 25 °C. Moreover, the pathogenicity of these strains fluctuated greatly by inducing rotting lesions on the pear fruits ranging from 1.5 mm to 34.0 mm in diameters at 5 dpi (Supplemental Table S3).

In this study, a novel mycovirus, BdRV1, were identified from a hypovirulent strain YZN115 of an important plant pathogenic fungus B. dothidea. The genome of BdRV1 consists of five dsRNAs with each dsRNA containing a single ORF. The proteins P1-P4 encoded by BdRV1 shared the highest aa sequence identities of 43–55% with the corresponding proteins encoded by AfuTmV-1, a virus infecting the human pathogenic fungus A. fumigatus (Kanhayuwa et al., 2015). The 50 - and 30 -terminal sequences of dsRNAs of BdRV1 were highly similar to the corresponding sequences of AfuTmV-1. In the most cases, the conserved terminal sequences of virus genomic RNAs play important roles in the package of viruses (Anzola et al., 1987; Wei et al., 2003). Then, the two viruses might have similar replication strategy. Additionally, the motif 6 of RdRp of both BdRV1 and AfuTmV-1 contain a GDN triad (catalytic residues), which has been identified in -ssRNA mycovirus SsNSRV-1 (Liu et al., 2014), but replaced by G/SDD in dsRNA and þssRNA viruses (Supplemental Fig. S1B). The dsRNA5 of BdRV1 could be detected in all single-conidium strains and other 13 strains. This result revealed that the dsRNA5 is an essential segment for BdRV1. However, the protein P5 encoded by dsRNA5 had no similarities to any known proteins in NCBI data. Therefore, further studies to elucidate the function of P5 in the BdRV1 are warranted. Therefore, despite that BdRV1 has the overall low sequence identity with AfuTmV-1 and one more dsRNA, the presence of the same domains and structural motifs in the corresponding proteins of encoded by both BdRV1 and AfuTmV-1 strongly support that that BdRV1 was a novel penta-segmented dsRNA virus and closely related to AfuTmV-1. The proline and alanine rich protein (PArp) was initially described in the Phlebiopsis gigantean large virus 1 (Kozlakidis et al., 2009). Some animal viruses can utilize a protein scaffold containing proline-rich “late” domain motifs to mediate proteinprotein interactions, interact with membrane components, and act as scaffold proteins anchoring replication complexes (Freed, 2002; Spear et al., 2010). It was suggested that PASrp encoded by RNA 4 of AfuTmV-1 was associated with the dsRNA genome of the virus and/or functioned in viral coating in an unconventional manner (Kanhayuwa et al., 2015). In this study, PMF and electrophoresis analyses revealed that a PASrp-like protein encoded by RNA4 of BdRV1 and the five genomic dsRNAs of BdRV1 could be recovered from pellets obtained after sucrose gradient differentiation combined with ultracentrifugation. Moreover, the results were confirmed by ELISA and RT-PCR. The findings indicated that virion-like particles (VLPs) were purified from the mycelia infected

Fig. 5. Reactivity of polyclonal antibody against P4 of BdRV1 in Western blot and indirect ELISA. (A) SDS-PAGE and the Western blot analysis of the protein P4 from purified BdRV1 preparation and total proteins of strains YZN115, JNT1111 and the derivative sub-strain Y15-J111-1 from strain JNT1111 in the hyphal contact with YZN115. The protein P4 was marked by the arrowhead. (B) Reactivity of PAB-p4 to strains YZN115 and JNT1111 in indirect ELISA. The data represented the mean absorbance values at a wave length of 450 nm of five replicates. The wells coated with skim milk were used as negative control.

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Fig. 6. Colony morphology and virulence on the shoots and fruits of pear and apple of strains YZN115 and JNT1111 and three derivate sub-strains. (A) Colony morphology in PDA medium (25 °C, 3d and 8d). (B) Shoots of pear (Pyrus pyrifolia cv. Hohsui) and apple (Malus domestica cv. Fuji) wound-inoculated with colonized plugs of YZN115 and JNT1111, and their sub-strains at 12 days post inoculation. (C) Fruits of apple (M. domestica cv. Fuji) and pear (P. bretschneideri cv. Huangguan) wound-inoculated with colonized plugs of YZN115 and JNT1111, and their sub-strains at 5 days post inoculation.

by BdRV1, which was similar to the finding for AfuTmV-1 (Kanhayuwa et al., 2015). However, in efforts in ISEM tests using antibody against the P4, there were no VLPs labeled with the antibody, which suggested that BdRV1 was unlikely to form classical virus particles. However, further studies will be necessary to answer the questions how the protein conjugated with the viral dsRNAs and whether BdRV1 presented in a form of unusual ribonucleoprotein as the case of AfuTmV1 (Kanhayuwa et al., 2015). Asexual conidia play important roles in the infections of the pathogenic B. dothidea. As other reported mycoviruses (Pearson et al., 2009), BdRV1 was easily transmitted into the progenies by asexual conidia. However, the hypervirulence of B. dothidea strains

conferred by BdRV1 may limit the expansion of the BdRV1infected B. dothidea strains. Importantly, the virus could be transmitted into some other strains by mycelium contacts despite that the vegetative incompatibility between different fungal strains could prevent the transmission of mycoviruses among different strains through hyphal contact or anastomosis (Nuss, 1992). In consistence, the virus widely distributes in the B. dothidea stains collected from different locations. In order to obtain a virus-free strain of YZN115, approaches, including picking hyphal tip, protoplast regeneration, and chemotherapy using ribavirin were employed to eliminate the BdRV1 from strain YZN115, but were unsuccessful. Although we could not compare the biological characteristics of virus-infected and virus-

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Table 2 The mycelium growth rates on PDA and virulence of BdRV1-free and BdRV1-infected strains of B. dothidea. Strainsn

JNT1111 YZN115 Y15-J111-1 Y15-J111-2 Y15-J111-3

Growth rate (mm/d)

42.8 7 0.4ae 16.3 7 2.5b 3.17 0.7c 8.5 7 2.6c 6.17 0.4c

Fruitsa lesion diameter (mm)b

Shootsc canker length (mm)d

P. bretschneideri

M. domestica

P. communis

P. pyrifolia

M. domestica

36.8 7 2.3a 11.17 0.4b 0.0 7 0.0c 10.5 7 0.7b 0.0 7 0.0c

68.3 76.3a 0.0 70.0b 0.0 70.0b 0.0 70.0b 0.0 70.0b

6.1 70.5a 0.0 70.0b 0.0 70.0b 0.0 70.0b 0.0 70.0b

27.2 75.6a 0.2 70.2b 0.0 70.0b 0.6 70.6b 0.1 70.1b

12.4 7 1.1a 0.0 7 0.0b 0.0 7 0.0b 0.0 7 0.0b 0.0 7 0.0b

n JNT1111 (BdRV1-free) and YZN115 (BdRV-infected) are wild strains as a negative control. Y15-J111-1, Y15-J111-2 and Y15-J111-3 are representative derivative substrains from JNT1111. a Fruits of pear (P. bretschneideri cv. Huangguan) and apple (M. domestica cv. Fuji.) were used in this test. b Data were measured at 5 dpi and calculated using lesion diameters of 8 inoculation sites. c Shoots of pear (P. pyrifolia cv. Nijisseiki, and P. communis cv. Lecounte) and apple (M. domestica cv. Fuji) were used in this test. d Data were calculated using canker length of 12 inoculation sites on shoots of detached shoots. e Letter indicate a significant difference (Po 0.05) among strains of B. dothidea according to the Student t test.

free YZN115 strains to give a direct support that BdRV1 was responsible for the hypervirulence of B. dothidea YZN115, BdRV1 reduced growth rate and virulence of some other strains derived from co-culture with YZN115 showed the association of the virus with the hypervirulence of B. dothidea. These characteristics permitted that the virus could be used as a potential biological agent for the controlling of diseases caused by B. dothidea (Nuss, 1992). Finally, sexual spores of B. dothidea are rarely observed in natural or artificial conditions (Slippers and Wingfield, 2007; Tang et al., 2012; Yan et al., 2013; Zhai et al., 2014). Whether the virus could be transmitted to the progenies of B. dothidea by sexual spores reminds to be elucidated. In conclusion, this study characterized a fungal dsRNA virus (BdRV1) in B. dothidea. BdRV1 can confer hypovirulence to the host B. dothidea and could be successfully transmitted via asexual spores and hyphal contact. BdRV1 has a close phylogenetic relationship with AfuTmV-1. They would be constituted a possibly new family which was an intermediate position between ssRNA and dsRNA viruses.

Acknowledgements This work was supported financially by the Chinese Ministry of Agriculture, Industry Technology Research Project (Grant nos.: 201203034-02 and 200903004-05) and the earmarked fund for Pear Modern Agro-industry Technology Research System (CARS29-10). The authors thank Professor Yangdou Wei, University of Saskatchewan, Canada, for his kind suggestions during the study.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://doi:10.1016/j.virol.2016.03.012.

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