A Novel Virus in the Family Hypoviridae from the Plant Pathogenic Fungus

Virus Research 174 (2013) 69–77

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Virus Research

jo urnal homepage: www.elsevier.com/locate/virusres

A novel virus in the family Hypoviridae from the plant pathogenic fungus

Fusarium graminearum

a b a a,∗ a,∗∗

Shuangchao Wang , Hideki Kondo , Liang Liu , Lihua Guo , Dewen Qiu

Key Laboratory of Integrated Pest Management in Crops, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences,

No. 12 Zhongguancun South Street, Beijing 100081, China

Institute of Plant Science and Resources (IPSR), Okayama University, Kurashiki, Okayama 710-0046, Japan

a r t i c l e i n f o a b s t r a c t

Article history: A double-stranded (ds) RNA element, sized at approximately 13 kb pairs, was puriﬁed from a ﬁeld isolate,

Received 19 December 2012

HN10, of Fusarium graminearum. The coding strand of the dsRNA was 13,023 nucleotides (nt) long (exclud-

Received in revised form 2 March 2013

ing the 3 poly(A) tail) and was predicted to contain two discontiguous open reading frames (ORF A and

Accepted 5 March 2013

ORF B). The 5 proximal ORF A of 531 nt encoded a protein of 176 amino acids (aa), and a BLAST search

Available online 14 March 2013

showed it to be similar to the putative papain-like protease domains encoded by Valsa ceratosperma

hypovirus 1 (35% identity) and Cryphonectria hypovirus 4 (CHV4) (31% identity). The 3 proximal ORF

Keywords:

B of 11,118 nt encoded a large polyprotein with three conserved domains, including papain-like pro-

dsRNA

Mycovirus tease, RNA-dependent RNA polymerase and RNA helicase domains. The polyprotein shared signiﬁcant

Hypoviridae aa identities with CHV1 (32%) and CHV2 (32%). Both the genome organization and phylogenetic analysis

Fusarium graminearum suggested that the characterized RNA represented a novel hypovirus, designated “Fusarium graminearum

Fusarium head blight hypovirus 1 (FgHV1)”, which was closely related to CHV1 and CHV2 in the Hypoviridae family. Elimination

FgHV1 of the virus resulted in no dramatic phenotypic alteration of the fungus.

1. Introduction CHV3 and CHV4 (Nuss and Hillman, 2011) that are phylogeneti-

cally related to the viruses in the plant-infecting family Potyviridae

Fungal viruses (mycoviruses) exist in all major groups of fungi. (Koonin et al., 1991) but distinguished in genome organization.

The number of known mycoviruses has been increasing rapidly The genomes of hypoviruses range from 9 to 13 kilobase (kb)

particularly due to extensive searches of plant pathogenic fungi in size and contain conserved domains of a papain-like protease,

(Ghabrial and Suzuki, 2009). Mycoviruses generally show asymp- an RNA-dependent RNA polymerase (RdRp) and an RNA helicase

tomatic infections, but a few viruses cause severe symptoms (Hillman and Suzuki, 2004). The identities of virus strains and

including hypovirulence (reduced virulence). Many mycoviruses species determine how severely symptoms are induced in the host

contain double-stranded (ds) RNA genomes, while some have fungus. CHV1 has been used for biological control of chestnut blight

plus-sense single-stranded (ss) RNA genomes. Viruses with ssRNA in Europe and the USA. The genome of CHV1 is 12.7 kb in size

genomes are grouped into ﬁve families: Hypoviridae, Narnaviridae, and has two open reading frames (ORF A and ORF B) divided by

Barnaviridae and Alphaﬂexiviridae (genera Botrexvirus and Sclero- the pentanucleotide UAAUG (Shapira et al., 1991) that act as the

darnavirus) and Gammaﬂexiviridae (King et al., 2011). Among these translation termination/reinitiation facilitator (Guo et al., 2009).

fungal viruses, the family Hypoviridae has contributed to enhancing Infection with the prototype hypovirus CHV1/EP713 produced

our understanding of fungal virus replication and symptom induc- drastically reduced virulence, sporulation and pigmentation, and

tion, virus molecular diversity and biocontrol of its fungal host, interrupted sexual reproduction of the fungal host (Anagnostakis,

the chestnut blight fungus Cryphonectria parasitica (Hillman and 1984). CHV1 has been studied in depth and the CHV1/C. parasi-

Suzuki, 2004; Nuss, 2005, 2011; Anagnostakis, 1982). tica system has served as a model for exploring mycovirus–fungus

The family Hypoviridae contains 4 members isolated from the interactions including the mechanisms underling fungal patho-

chestnut blight fungus, Cryphonectria hypovirus 1 (CHV1), CHV2, genesis (Nuss, 2005; Milgroom and Cortesi, 2004). CHV2/NB58

has a genome of 12.5 kb and also contains two ORFs (Hillman

et al., 1994). CHV2/NB58 reduces fungal virulence, development

and fecundity (Hillman et al., 1992). The 9.8-kb CHV3/GH2 con-

∗

Corresponding author. Tel.: +86 13121205315; fax: +86 10 82109562.

∗∗ tains a single ORF and can also reduce fungal virulence but does

Corresponding author.

not have a substantial impact on pigmentation, conidiation, or lac-

E-mail addresses: [email protected] (L. Guo), [email protected]

case expression (Fulbright, 1984; Smart et al., 1999; Hillman et al.,

(D. Qiu).

70 S. Wang et al. / Virus Research 174 (2013) 69–77

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2000; Yuan and Hillman, 2001). CHV4/SR2, with a 9.1 kb genome medium (CM) at 25 C. Mycelial plugs were stored in 25% glycerol

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that contains a single ORF, has little effect on fungal virulence frozen at −80 C.

and colony morphology (Linder-Basso et al., 2005). Recently, two

novel hypoviruses were isolated from different phytopathogenic 2.2. dsRNA detection and puriﬁcation

fungi and were designated Sclerotinia sclerotiorum hypovirus 1

(SsHV1/SZ150, 9.5 kb genome) and Valsa ceratosperma hypovirus Mycelial plugs of strain HN10 were placed onto a PDA (potato

1 (VcHV1/MVC86, 9.5 kb genome) (Xie et al., 2011; Yaegashi et al., dextrose agar) plate overlaid with cellophane membranes and cul-

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2012). These viruses contain single ORFs and are closely related tured for 4 days at 25 C. Subsequently, mycelial mass was collected

to each other and to CHV3 and CHV4. Yaegashi et al. (2012) and used for dsRNA extraction using the cellulose chromatography

proposed that these hypoviruses should be classiﬁed into two gen- (Sigma–Aldrich, Dorset, England) method as previously described

era, “Alphahypovirus” (CHV1 and CHV2) and “Betahypovirus” (the (Valverde, 1990). The puriﬁed dsRNA was digested with DNase I

remaining hypoviruses). and S1 nuclease (TaKaRa Bio Inc., Dalian, China) following the man-

Fusarium head blight (FHB; also known as scab) is a devastating ufacturer’s instructions. To further conﬁrm the nature of the dsRNA,

disease of wheat, barley and other small-grain cereals, and out- the extracted segment was incubated with 2 ␮g/ml of RNase A in

breaks in Europe, America, Canada and China during the 1990s low ionic strength buffer (0.1 × SSC [1 × SSC = 0.15 M NaCl, 0.015 M

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have made FHB a global problem (O’Donnell et al., 2004). FHB sodium citrate, pH 7.0]) for 30 min at 37 C (Vilches and Castillo,

attracts attention not only because it causes severe loss of grain 1997). The sample was electrophoresed on a 1% agarose gel and

yield and quality reduction but also because it raises animal and visualized using ethidium bromide staining.

human health concerns about mycotoxin contamination (Rocha

et al., 2005). Fusarium graminearum (teleomorph Gibberella zeae) is 2.3. Complementary DNA (cDNA) cloning and sequencing

the primary causal agent of FHB (O’Donnell et al., 2000). To date, few analysis

mycoviruses have been reported for the plant pathogenic fungus F.

graminearum (Theisen et al., 2001; Chu et al., 2002; Yu et al., 2009). cDNAs of the puriﬁed dsRNA were obtained by reverse

Fusarium graminearum virus 1 (FgV1), FgV2, FgV3 and FgV4 were all transcription polymerase chain reaction (RT-PCR). The dsRNA

identiﬁed from isolates of Fusarium species in Korea. Among them, mixed with tagged random primers-dN6 (5 -GACGTCCAGATCGCG-

◦

FgV1 is the only one that is associated with deliberation of host vir- AATTCNNNNNN-3 ) was denatured at 95 C for 10 min and

ulence. FgV1 has a (+) ssRNA genome of about 6.6 kb and is encased immediately chilled on ice for 5 min. The reverse transcription

in pleomorphic vesicles. Its genome expression strategy is similar was conducted using M-MLV Reverse Transcriptase (Promega,

to plant-infecting ssRNA viruses in the utilization of subgenomic USA) according to the manufacturer’s instructions. The result-

mRNAs for expression of downstream ORFs. However, based upon ing random cDNAs were ampliﬁed using a single speciﬁc primer

its RdRp sequence, FgV1 is evolutionarily more closely related to (5 -GACGTCCAGATCGCGAATTC-3 ) and PrimeSTAR HS DNA Poly-

hypoviruses (Chu et al., 2002; Kwon et al., 2007). The FgV2 genome merase (TaKaRa) on a Thermal Cycler (Bio-Rad, Hercules, CA, USA),

has been partially sequenced and is contained in virus-like particles following the manufacturer’s protocols. The PCR product was sep-

in the cytoplasm of the host cells (Chu et al., 2004). FgV3 and FgV4 arated on a 1% agarose gel and puriﬁed using a gel extraction kit

are both found in strain DK3, which shows mixed infection with (Sigma). The products were ligated to the PMD18-T vector and

mycoviruses (Yu et al., 2009). FgV3 is closely related to members transformed into Escherichia coli strain DH5␣ (TaKaRa). Positive

of the families Totiviridae and Chrysoviridae, whereas FgV4 is most clones were selected for sequencing using the Beijing Genomics

likely a member of the family Partitiviridae. Darissa et al. (2011) Institute service (BGI, Shenzhen, China).

screened for dsRNA mycoviruses from F. graminearum isolates from The gap sequences between the cDNAs were also determined by

China and found a virus that may be a novel member of the family RT-PCR. The puriﬁed dsRNA mixed with tagged random primers-

◦

Chrysoviridae. dN6 was heat-denatured at 95 C for 10 min and chilled on ice

In this study, we report molecular and biological characteriza- for 5 min. Subsequently, the dsRNA was reverse transcribed using

tion of a novel hypovirus with a distinct 2-ORF genome structure PrimerScript Reverse Transcriptase (TaKaRa). The enzymatic reac-

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from Fusarium graminearum strain HN10 and name it “Fusarium tion was incubated at 42 C for 60 min and stopped by heating

◦

graminearum hypovirus 1 (FgHV1)”. We also report its closer rela- at 70 C for 10 min. The resulting cDNA was used as the tem-

tionship to CHV1 and CHV2 than to CHV3 and CHV4. plate for PCR using PrimerSTAR GXL DNA Polymerase (TaKaRa),

which can achieve the ampliﬁcation of long fragments. Pairs of

2. Materials and methods primers to PCR amplify the sequences of concatenated cDNAs were

designed based on the sequences obtained for the random cDNAs

2.1. Fungal isolates and culture conditions (see supplementary Table S1). PCR products were puriﬁed using

a gel extraction kit (Sigma), cloned and sequenced as described

Samples of wheat infected with Fusarium spp. were collected above.

from different ﬁelds in 7 provinces covering most of the wheat Supplementary material related to this article found, in the

producing areas in China. We recovered Fusarium spp. from the online version, at http://dx.doi.org/10.1016/j.virusres.2013.03.002.

diseased seeds or glumes as described (Chu et al., 2002). F. gramin- Three methods were used to determine the terminal sequences

earum strain HN10 was isolated from diseased seeds of wheat of the dsRNA element. A classic 5 -RACE (rapid ampliﬁcation of

infected with Fusarium spp. collected in the Henan province of cDNA ends) protocol using terminal deoxynucleotidyl transferase

China. Strain HN10-11F is a virus-free strain derived from strain was performed as described by Suzuki et al. (2004). A classic

HN10 by protoplast isolation. F. graminearum strain PH-1 cells 3 -RACE protocol using an adaptor-linked oligo-dT primer, was per-

were transformed using plasmid pUCATPH with the bacterial formed as described by the instructions for the TaKaRa 3 -Full RACE

hygromycin B resistance gene (hph, hygromycin B phosphotrans- Core Set Ver.2.0 (TaKaRa). We carried out 3 RNA Ligase-Mediated

ferase) (Lu et al., 1994) for a viral transmission assay (Hou et al., RACE (RLM-RACE) as described by Xie et al. (2006) to ascertain

2002). The resulting virus-free hygromycin-B-resistant transfor- the 3 -terminal sequences of each strand of the dsRNA element.

mant PH-1-2 was used for studies using hyphal anastomosis. Strain Speciﬁc primers for ampliﬁcation of the 5 -end (19R and AR) and

PH-1-2V, which was infected with the virus through hyphal anasto- the 3 -end cDNAs (EF and EF-2) are listed in supplementary Table

mosis, is isogenic to PH-1-2. All strains were cultured on complete S1. The ampliﬁed cDNA fragments were ligated into the pMD18-T

S. Wang et al. / Virus Research 174 (2013) 69–77 71

vector, transformed into E. coli DH5 and sequenced as described SAS 8.0 program. Treatment means were compared using the least

above. Resulting sequences were assembled and analyzed using signiﬁcant difference (LSD) test at the P = 0.01 level.

DNAMAN software (Lynnon Biosoft, Quebec, Canada). Homology

searches were carried out using the National Center for Biotech-

2.8. Nucleotide sequence accession numbers

nology Information (NCBI) Blast program.

The complete nucleotide sequence of the dsRNA isolated from

2.4. Phylogenetic analysis

strain HN10 has been deposited in GenBank under accession no.

KC330231.

Phylogenetic tree construction was based on a

maximum-likelihood (ML) method as described previously

3. Results

(Chiba et al., 2011; Kondo et al., 2013). Details of the proce-

dure are as follows. The deduced amino acid sequences of the

3.1. dsRNA in F. graminearum strain HN10

hypovirus proteins or conserved domains in the polyproteins

were aligned using MAFFT version 6 with the default parameters

dsRNA extraction from Fusarium spp. isolates collected in differ-

(Katoh and Toh, 2008). All regions having an alignment gap

ent parts of China revealed that many strains contained different

were completely removed using MEGA version 4.02 software

sizes of virus-like dsRNAs. We found 18 out of 195 samples to

(Tamura et al., 2007). Selection of the best-ﬁt model for a data set

be virus-like dsRNA positive (data not shown). One of the ﬁeld

was performed using ProtTest (Abascal et al., 2005). Maximum

isolates, HN10, which had been isolated from Henan, the high-

likelihood phylogenetic trees were generated using PhyML 3.0

est wheat production province in China, was chosen for further

(Guindon et al., 2010). The branch support values were esti-

analysis. The HN10 isolate was identiﬁed as F. graminearum by

mated using the approximate likelihood ratio test (aLRT) with a

PCR ampliﬁcation of the EF-1a fragment as described (O’Donnell

Shimodaira–Hasegawa-like (SH-like) algorithm (Anisimova and

et al., 2000) (see supplementary Table S1 for primer sequences

Gascuel, 2006). Phylogenetic trees were visualized using Figtree

used) (data not shown). Agarose gel electrophoresis showed that

(version 1.3.1) (http://tree.bio.ed.ac.uk/software/ﬁgtree/).

the strain carried a single dsRNA element sized at more than

10 kbp (Fig. 1A). The segment was conﬁrmed to be dsRNA in

2.5. Curing Fusarium graminearum strain HN10 of virus and

nature based on its resistance to DNase I and S1 nuclease and

virus transmission

susceptibility to a low salt concentration digestion with RNase A

(Fig. 1A).

The protoplast isolation and regeneration method was used

to cure the virus from the host strain. Protoplasts were prepared

according to the method of Hou et al. (2002). The protoplasts 3.2. Nucleotide sequence and genome organization of the F.

derived from HN10 were screened to determine whether they car- graminearum dsRNA virus-like agent

ried the virus. The virus was transmitted into another virus-free F.

graminearum strain, PH-1-2, with the hygromycin resistance gene The complete nucleotide sequence of the dsRNA of over 10 kbp

(hph), using the hyphal anastomosis method. Mycelial plugs of was determined by sequencing of random-primed cDNA, RT-PCR

HN10 and PH-1-2 were placed onto the surface of a PDA plate at a and RACE clones. One hundred and thirty-two clones were obtained

◦

distance of 1.5 cm and co-cultured at 25 C. After the hyphae had and fully sequenced in both directions. Every base was determined

intermingled, mycelial plugs were taken from the margin of the by sequencing of 3–24 independent clones (Supplementary Fig.

recipient PH-1-2 strain and transferred onto a PDA plate containing S1A). The ampliﬁcation of a speciﬁc PCR product using an oligo-dT

hygromycin B at a concentration of 100 g/ml. The mycelial plugs primer demonstrated that a poly(A) tail existed at the 3 terminus

that could grow on the hygromycin-B-containing PDA were further of its plus strand. The complete genomic sequence (coding strand)

used for dsRNA detection. In addition, the virus was introduced was 13,023 nt, excluding the 3 -terminal poly(A) tail, in length. The

back into the virus-free strain HN10-11F using the same method. genome organization is shown in Fig. 1B. The coding strand of the

The absence or existence of dsRNA agents was detected using dsRNA encoded two ORFs (ORFs A and B). We also found another

Northern dot blot analysis and RT-PCR using a probe and primer four smaller ORFs (300–500 nt in length) in the central region of

pair for the 8R and 8F regions (supplementary Table S1). both the plus and minus genomic strands (Supplementary Fig. S1B).

The potentially encoded proteins of these small ORFs did not have

2.6. Impact of the virus on host biological properties any detectable similarity to the NCBI protein database (data not

shown). Thus, these smaller ORF candidates were not examined

Mycelial growth, conidiation, virulence and toxin production of further.

strain HN10 and its protoplast derivate HN10-11F were assessed. Supplementary material related to this article found, in the

Growth rates on PDA and CM plates were examined by measur- online version, at http://dx.doi.org/10.1016/j.virusres.2013.03.002.

◦

ing colonial diameters after culturing for 72 h at 25 C. Conidial The two major ORFs, A and B, were separated by 384 nt con-

production in 5-day-old carboxymethyl cellulose (CMC) broth was taining eight mini-cistrons. ORF A, beginning at AUG (nt positions

determined on blood count plates. To assess virulence, wheat 511–513) and terminating at UAG (nt positions 1039–1041), was

head infections were conducted as described (Gale et al., 2002; predicted to encode a 176-aa protein with a molecular mass of

Kang and Buchenauer, 1999), and the assay for deoxynivalenol 20 kDa. No putative conserved domains were detected in the

(DON) production was executed as described (Bluhm et al., 2007; ORF A protein (p20) using the conserved domain search program

Seong et al., 2006). In addition, virus-infected strain PH-1-2 V was on the NCBI web site. A BLASTp search using the deduced aa

compared with its isogenic strain PH-1-2 according to the above sequence of ORF A showed that it had sequence identities with the

aspects. putative papain-like protease domain of a polyprotein encoded

by VcHV1/MVC86 (E-value = 4e−23; identities = 65/187 = 35%) and

2.7. Data analysis CHV4/SR2 (E-value = 9e−17; identities = 56/181 = 31%). ORF B (nt

positions 1426–12,543) encoded a large polyprotein of 421 kDa that

Each experiment included at least 3 replicates, and experimen- was predicted to consist of 3705 aa. The deduced ORF B protein con-

tal data were subjected to analysis of variance (ANOVA) using the tained three conserved domains including papain-like proteinase

72 S. Wang et al. / Virus Research 174 (2013) 69–77

Fig. 1. (A) A dsRNA element in Fusarium graminearum strain HN10. The dsRNA fraction extracted from F. graminearum strain HN10 was electrophoresed in a 1% agarose

gel and visualized under UV light after staining with ethidium bromide. Lane M, DNA marker; Lane 1, dsRNA sample after treatment with both RNase-free DNase I and S1

nuclease. Lane 2, dsRNA sample incubated with RNase A in low ionic-strength buffer. (B) Schematic representation of the genomic organization of Fusarium graminearum

hypovirus 1 (FgHV1/HN10). The FgHV1 genome is 13 kb long and contains two ORFs, ORF A (p20) and ORF B (polyprotein), and a putative internal untranslated region (UTR,

384 nt) separating the two ORFs. Thick black lines indicate 5 -, 3 - and internal UTRs. The black boxes in ORF B represent conserved domains including papain-like protease

(Pro), RNA-dependent RNA polymerase (RdRp) and RNA helicase (Hel) domains. A possible cleavage site for the cis-acting proteinase is marked by the curved arrow and

has not yet been proven by experiments. A poly(A)-tail at the 3 -end of the coding strand is represented as A(n). (C). A maximum likelihood (ML) tree of FgHV1/HN10 and

other hypoviruses based on full length amino acid sequences of the viral polyproteins was constructed using PhyML 3.0. Gaps in multiple alignment were removed manually

using MEGA4 and the resulting cured alignment was subjected to phylogenetic analysis (data not shown). Numbers at the nodes represent aLRT values derived using an

SH-like calculation. Accession numbers are CHV1/EP713, Cryphonectria hypovirus 1-EP713 (accession no. M57938); CHV2/NB58, Cryphonectria hypovirus 2-NB58 (L29010);

CHV3/GH2, Cryphonectria hypovirus 3-GH2 (AF188514); CHV4/SR2, Cryphonectria hypovirus 4-SR2 (AY307099); SsHV1/SZ150, Sclerotinia sclerotiorum hypovirus 1-SZ150

(JF781304); VcHV1/MVC86, Valsa ceratosperma hypovirus 1-MVC86 (AB690372) and FgV1/DK21, Fusarium graminearum virus 1-DK21 (AY533037).

(Peptidase C7 [pfam01830]; E-value = 1e−18), RdRp (DUF3525 similar to those of hypoviruses. Thus, we presumed that this

[pfam12039]; E-value = 3e−21) and RNA helicase (DEXDc virus-like dsRNA element was a replication intermediate of a novel

[cd00046]; E-value = 1e−4) domains. A homology search with mycovirus closely related to ssRNA mycoviruses in the genus

the sequence of the polyprotein encoded by ORF B revealed that it Hypovirus, tentatively named “Fusarium graminearum hypovirus

had sequence similarities to the polyproteins of CHV1/EP713 (ORF 1 (FgHV1/HN10)”.

B) (E-value = 0.0; identities = 752/2427 = 31%), CHV2/NB58 (ORF

−15 −11

B) (0.0; 493/1550 = 32%), CHV3/GH2 (8e , 2e ; 63/196 = 32%, 3.3. Sequence similarities and phylogenetic analysis of

−8

205/976 = 21%), CHV4/SR2 (1e ; 166/781 = 21%), VcHV1/MVC86 FgHV1/HN10 and other hypoviruses

−7

(1e , 0.086, 0.15; 109/466 = 31, 31/100 = 31, 62/227 = 27%),

−7

SsHV1/SZ150 (3e ; 146/635 = 23%) and possibly to the ssRNA To deﬁne the relationships between FgHV1/HN10 and other

mycovirus FgV1/DK21 ORF1 (0.002; 33/124 = 27%). We also found hypoviruses, including CHV1/EP713, CHV2/NB58, CHV3/GH2,

several matches including potential hypovirus or related ssRNA CHV4/SR2, SsHV1/SZ150, VcHV1/MVC86 and FgV1/DK21, phylo-

mycovirus sequences from Alternaria alternata (GenBank acces- genetic analyses were performed based on the viral polyproteins.

−179

sion HE579694; E-value = 2e ; identities = 356/1039 = 34%) The maximum likelihood (ML) tree of the polyproteins suggested

(Feldman et al., 2012), Trichoderma asperellum (AFR77082, that FgHV1 is more closely related to CHV1/EP713 and CHV2/NB58

−42 −8

AFR77083; 1e , 3e ; 100/262 = 31%, 47/158 = 30%), Agaricus than to CHV3/GH2, CHV4/SR2, SsHV1/SZ150 and VcHV1/MVC86

−13

bisporus (BAM36408; 3e ; 78/278 = 28%), Rosellinia necatrix (Fig. 1C). Notably, the ﬁrst 120 nt of the 5 UTR and the last 120 nt of

−5

(BAM36408; 3e ; 30/108 = 28%) (Yaegashi et al., 2013) and the 3 UTR of FgHV1/HN10, excluding the poly(A) tail, shared sev-

grapevine plant (GU108591; 0.007; 33/110 = 30%) (Al Rwahnih eral sequence stretches with those of CHV1/EP713 and CHV2/NB58

et al., 2011). The 5 - and 3 -untranslated regions (UTRs) were (Supplementary Fig. S2A and B). In addition, the similarities

determined as 510 and 480 nts, respectively, excluding the 3 - between FgHV1/HN10 and CHV1 in the 5 UTR were observed in

poly A tail. The genome organization of the dsRNA element was the presence of multiple upstream AUG codons (FgHV1/HN10, six

S. Wang et al. / Virus Research 174 (2013) 69–77 73

Fig. 2. Multiple alignment (A) and phylogenetic tree (B) of the putative papain-like protease (Pro) domains of FgHV1/HN10, other hypoviruses and a potyvirus. (A) Pro domain

sequences were aligned using the MAFFT program. Positions of the catalytic Cys and His residues and a possible cleavage site (Gly) are highlighted. Asterisks and colons

indicate conserved or semi-conserved amino acid residues, respectively. (B) A maximum likelihood (ML) tree of the Pro domain sequences was constructed using PhyML 3.0.

Gaps in the multiple alignment were removed manually using MEGA4 and the resulting cured alignment was subjected to phylogenetic analysis. Accession numbers are as

in Fig. 1 with the addition of PPV, plum pox virus-NAT (D13751). Numbers at the nodes represent aLRT values derived using an SH-like calculation.

74 S. Wang et al. / Virus Research 174 (2013) 69–77

and CHV1, seven) and structural complexity with numerous stem-

loop structures predicted using Mfold version 2.3 (Zuker, 2003)

(Web server URL: http://mfold.rna.albany.edu/) (Supplementary

Fig. S2C).

Supplementary material related to this article found, in the

online version, at http://dx.doi.org/10.1016/j.virusres.2013.03.002.

All members of the Hypoviridae family encode three domains:

papain-like protease (Pro), RNA-dependent RNA polymerase

(RdRp), and RNA helicase (Hel) domains. A Pro domain (133–226 aa)

of FgHV1 was located at the N-terminal end of the ORF B polypro-

tein of FgHV1/HN10. Although no further Pro domains could be

detected in our motif search, the C-terminal part of the ORF A pro-

tein of this virus showed overall similarity to the Pro domains of

some hypoviral proteins: The terminal sequence of the papain-like

protease is cysteine-rich and two strictly conserved residues (Cys

and His) are required for its autoproteolytic activity (Hillman et al.,

1994; Koonin et al., 1991; Smart et al., 1999). Multiple alignments

of the putative Pro domains of the ORF A and ORF B proteins of

FgHV1/HN10 and those of other selected viruses detected two key

104 154

residues (at the Cys and His positions in the ORF A protein

140 192

and Cys and His in the ORF B protein) (Fig. 2A). A puta-

226

tive polyprotein cleavage site (position Gly ) was deduced for

the ORF B papain-like protease of FgHV1/HN10 (Fig. 2A), not far

downstream from the two catalytic residues, similar to previously

reported hypovirus proteases (Yuan and Hillman, 2001). In con-

trast, the conserved residue at the cleavage site for ORF A putative

176

Pro (position Gly ) is located just at the C terminus of this protein

(Fig. 2A). Therefor, we speculated that ORF A-encoding p20 might

not function as a papain-like protease while possessing the puta-

tive Pro domain. In comparison, ORF B-encoding p25 has features

characteristic of the hypovirus papain-like protease. Phylogenetic

analysis based on the putative Pro domains among FgHV1/HN10,

other hypoviruses, FgV1/DK21 and a plant potyvirus, plum pox virus

(PPV) were performed. The ML tree based on the papain-like pro-

tease motif of these viruses indicated that the ORF B Pro domain

of FgHV1/HN10 was more closely related to those of CHV3/GH2

and CHV1/EP713-A (p29) than to others, and in contrast, the ORF

A putative Pro domain was clustered with those of CHV4/SR2 and

VcHV1/MVC86 (Fig. 2B).

The RdRp and RNA helicase (Hel) domains of FgHV1/HN10 Fig. 3. Phylogenetic trees of the RNA-dependent RNA polymerase (RdRp) (A) and

RNA helicase (Hel) (B) domains of FgHV1/HN10, other hypoviruses and a potyvirus.

(RdRp, 2371–2773 aa; RNA helicase, 3210–3480 aa) were located

ML trees were constructed using PhyML 3.0. Gaps in the multiple alignments of the

at the C-terminal of the polyprotein encoded by ORF B. Multiple

RdRp and Hel domains (shown in Fig. S3) were removed manually using MEGA4 and

alignments showed that the RdRp and Hel domains were highly the cured alignments were subjected to phylogenetic analysis. Accession numbers

conserved among FgHV1/HN10 and other hypoviruses. There are are as in Figs. 1 and 2. Numbers at the nodes represent aLRT values derived using an

SH-like calculation.

several conserved amino residues in the RdRp domain consist-

ing of eight motifs (Koonin et al., 1991) (Supplementary Fig.

S3A). It is worth noting that an SDD tripeptide was highly con- CHV3/GH2, CHV4/SR2, SsHV1/SZ150 and VcHV1/MVC86 (Fig. 3B).

served among the previously characterized hypoviruses. However, Sequence alignment and phylogenetic analysis based on the RdRp

the ﬁrst amino acid of the corresponding tripeptide was a sub- or the Hel domain indicated that FgHV1/HN10 was most closely

2700

stituted Gly residue in FgHV1/HN10, the same as found in related to CHV1/EP713 and CHV2/NB58 but was distinct from

FgV1/DK21 and PPV (Supplementary Fig. S3A). The RdRp domain CHV3/GH2, CHV4/SR2, SsHV1/SZ150 and VcHV1/MVC86 in the

of FgHV1/HN10 shared higher sequence identities with those of genus Hypovirus.

CHV1/EP713 (32%) and CHV2/NB58 (31%), but lower identities Supplementary material related to this article found, in the

with those of CHV3/GH2 (20%), CHV4/SR2 (18%), SsHV1/SZ150 online version, at http://dx.doi.org/10.1016/j.virusres.2013.03.002.

(22%) and VcHV1/MVC86 (27%) (Table 1). The phylogenetic anal-

ysis based on the RdRp domain showed that FgHV1/HN10 was 3.4. Biological effect of FgHV1/HN10 on F. graminearum

more closely related to CHV1/EP713 and CHV2/NB58 than to

CHV3/GH2, CHV4/SR2, SsHV1/SZ150 and VcHV1/MVC86 (Fig. 3A). Two isogenic pairs of virus-free and carrying fungal strains

The Hel domain of FgHV1/HN10 shared the conserved motifs of were biologically compared. To eliminate FgHV1 from the HN10

the DExH box and the QRxGR box (x, any amino acid residue) fungal strain, we utilized single spore isolation. However, 252

that are characteristic motifs in the helicase superfamily 2 (Hall single asexual spore isolates tested were all found to be viral

and Matson, 1999) (Fig. S3B). The Hel domain in FgHV1/HN10 had dsRNA (FgHV1/HN10)-positive (data not shown). By protoplas-

higher identities to those in CHV1/EP713 (34%) and CHV2/NB58 ting/regeneration, we obtained one strain, HN10-11F, that was

(34%) than those of other hypoviruses (13–26%) (Table 1). The phy- FgHV1 free and served as an isogenic strain to HN10. For the

logenetic tree of the helicase domain also showed that FgHV1/HN10 second pair, through hyphal anastomosis F. graminearum strain

was more closely related to CHV1/EP713 and CHV2/NB58 than to PH-1-2V, with the hygromycin B resistance marker, was infected

S. Wang et al. / Virus Research 174 (2013) 69–77 75

Table 1

Nucleotide and amino acid identities between FgHV1/HN10 and other hypoviruses.

Identities to FgHV1/HN10

a a

Complete seq. 5 UTR 3 UTR ORF A ORF B Domain (aa%)

Length (nt) nt% Length (nt) nt% Length (nt) nt% (aa%) (aa%) Pro RdRp Hel

b d

CHV1/EP713 12,712 50 495 48 851 44 – 32 27/18 32 34

CHV2/NB58 12,507 49 487 58 828 46 – 32 – 31 34

CHV3/GH2 9799 47 369 52 805 43 – 24 37 20 24

CHV4/SR2 9149 46 193 47 409 42 31 19 – 18 13

SsHV1/SZ150 10,398 48 541 49 1010 45 – 22 – 22 25

VcHV1/MVC86 9543 48 378 48 342 36 35 23 – 27 26

FgV1/DK21 6621 49 53 36 46 49 – 21 – 28 21

The terminal 120 nt of the 5 - and 3 -UTR sequences were compared to the respective UTR of each virus.

No signiﬁcant identity was detected.

A short stretch of homology is seen between these sequences.

Identity to the protease domain of ORF A or ORF B of CHV1 is shown to the left and right of the slash, respectively.

with the virus. Virus-carrying strain HN10 was indistinguishable sites to nearby spikelets at the same speed as the virus-free strain

from its isogenic virus-free strain HN10-11F in colony morphology HN10-11F. After 14 days post inoculation (dpi), there was no sig-

(Fig. 4B). Strain HN10 had minor reductions in mycelial growth rate niﬁcant difference in the numbers of diseased spikelets per wheat

on CM plates (17.3% reduction, p < 0.01) and conidial production head invaded (n = 15) (Fig. 4E). DON is a trichothecene mycotoxin

(28% reduction, p < 0.01) (Fig. 4C and D). In a virulence assay, the produced by F. graminearum that shows a positive correlation with

virus-carrying strain HN10 was also able to spread from inoculation the virulence of F. graminearum. In addition, DON production by

Fig. 4. Biological impact of FgHV1 infection on Fusarium graminearum. (A) Detection of viral dsRNA using Northern dot blot (top row) and RT-PCR (middle row) of individual

F. graminearum isolates HN10-11F (virus-free), HN10 (virus carrying), PH-1-2 (virus-free) and PH-1-2V (virus infected). Lane M, DNA marker. To check RNA quality, the

ribosomal RNA pattern (28S and 18S) was observed after gel electrophoresis of the total RNA from each sample on a 1% agarose gel (bottom row). (B) Colony morphology

of HN10 and HN10-11F (virus-free) after 3 days of culture on complete medium (CM). (C and D) Comparison of the colonial diameter (C) and conidial production (D) of

virus-free and virus-carrying fungal strains after culture on CM for 3 days and CMC for 5 days. (E and F) Assessment of virulence (E) and mycotoxin DON production (F) on

wheat grain by virus-free and virus-carrying fungi after 14 days post inoculation. Bars (C–F) indicate standard deviations.

76 S. Wang et al. / Virus Research 174 (2013) 69–77

strains HN10 and HN10-11F were nearly identical, consistent with with CHV4 and VcHV1 (“Betahypovirus”) (Fig. 2). In addition, the

the results of the virulence assay (n = 15) (Fig. 4F). The comparative geographical distribution of hypoviruses belonging to the two lin-

results were conﬁrmed by comparisons between PH-1-2 and eages contributes to the likelihood of mycovirus recombination.

PH-1-2V according to the same characteristics (Fig. 4C–F). In Members of the “Alphahypovirus” and “Betahypovirus” groups have

addition, after we re-introduced the virus into virus free strain both been detected in Asia. CHV1 and CHV2 were both found in

HN10-11F, mycelial growth demonstrated the same comparative Asia, SsHV1 in China and VcHV1 in Japan (Peever et al., 1997, 1998;

results (data not shown). Therefore, FgHV1/HN10 infection had Xie et al., 2011; Yaegashi et al., 2012). Thus, a recombination event

mild or no effects on fungal phenotypes including morphology, might have occurred in a doubly infected host fungus in Asia. Nev-

mycelial growth, conidiation and virulence or toxin production. ertheless, in considering the evolutionary history of FgHV1, it is

interesting to note that the Pro domain of FgHV1/HN10-B was more

4. Discussion closely related to that of CHV1/EP713-A (p29) than CHV1/EP713-B

(p48) and CHV2/NB58 (Fig. 2B). It is tempting to speculate the

In this study, we described molecular and biological charac- closest relatives between FgHV1 and CHV1 crossing the family

terization of the novel mycovirus FgHV1 isolated from the plant Hypoviridae. The characterization of FgHV1 will contribute to explo-

pathogenic fungus F. graminearum isolate HN10. FgHV1/HN10 is ration of the ecology and evolution of mycoviruses in the family

characterized by its latent infection in the host, two-ORF genome Hypoviridae.

structure and sequence similarity of encoded proteins to corre- FgHV1 infection caused mild or no impact on the phenotype and

sponding proteins of other hypoviruses (Fig. 1 and Table 1). The virulence of its host F. graminearum, which is different from infec-

similarities to hypoviruses extend to the long 5 UTR (510 nt) tions by the type strains of CHV1 (strain EP713) and CHV2 (strain

with six mini-cistrons and to the genome size. The complete NB58) (Fig. 4B). Virulence levels among different hypoviruses

nucleotide sequence of the virus genome is 13,023 nt (the longest vary considerably. Among reported hypoviruses, CHV1/EP713,

among hypoviruses), excluding the 3 -terminal poly(A) tail. The CHV2/NB58, and CHV3/GH2 infections reduce virulence of host

genome of FgHV1 contains two ORFs that are separated by fungi (Hillman et al., 1990, 1992, 1994; Fulbright, 1984; Smart

384 nt, a 2-discontiguous-ORF genome structure distinct from et al., 1999). SsHV1/SZ150 alone is not the primary causal agent for

other hypoviruses. Based on these results, it is hypothesized that hypovirulence of strain SZ150 since strains without its satellite-like

FgHV1 represents a novel mycovirus closely related to hypoviruses. small dsRNA show normal phenotype (Xie et al., 2011). CHV4/SR2

Currently, there are six hypoviruses assigned to the family and VcHV1/MVC86 have little effect on colony morphology and

Hypoviridae including four from C. parasitica (CHV1–4), one from virulence of their hosts (Linder-Basso et al., 2005; Yaegashi et al.,

S. sclerotiorum (SsHV1) and one from V. ceratosperma (VcHV1) 2012). In the case of CHV1, the two proteases p29 and p48 play piv-

(Nuss and Hillman, 2011; Xie et al., 2011; Yaegashi et al., 2012). otal roles in symptom induction and replication, but they are not

Yaegashi et al. (2012) suggested the existence of two lineages in responsible for virulence attenuation (Craven et al., 1993; Suzuki

the family Hypoviridae, one lineage named “Alphahypovirus” that et al., 2003; Sun et al., 2006; Deng and Nuss, 2008). The replication

includes CHV1 and CHV2, the other named “Betahypovirus” which enhancer activity of p29 may be a manifestation of its suppressor

consists of CHV3, CHV4, SsHV1 and VcHV1. The two members with activity against the host’s RNA silencing mechanism (host defense

non-Cryphonectria fungal hosts, SsHV1 and VcHV1, are both closely against infecting viruses) (Segers et al., 2007). Our phylogenetic

related to CHV3 and CHV4 and placed into one clade within the tree showed that the protease of FgHV1-ORF B (p25) clustered into

Hypoviridae family (Xie et al., 2011; Yaegashi et al., 2012). FgHV1 a clade with those of CHV1/EP713-A (p29) and CHV3/GH2 while the

from F. graminearum is another new member in the family Hypoviri- putative Pro domain of FgHV1-ORF A (p20) clustered with those of

dae. Notably, FgHV1, isolated from an F. graminearum strain in CHV4/SR2 and VcHV1/MVC86 (Fig. 2B). Whether the p25 and p20

China, is closely related to CHV1 and CHV2 (Table 1). The phyloge- act as suppressors of RNA silencing and contribute to FgHV1 RNA

netic analysis indicates that FgHV1 clusters into one clade together accumulation, as described for p29 (Segers et al., 2007), requires

with CHV1 and CHV2. No UDP-glucose/sterol glucosyltransferase future investigation.

domain, conserved in CHV3, CHV4, VcHV1 and SsHV1, was found

in FgHV1. So FgHV1 was assigned to “Alphahypovirus” together with Acknowledgments

CHV1 and CHV2 (Fig. 3 and Supplementary Fig. S3). Also the notion

that FgHV1 is more closely related to the Alphahypovirus is sup- This work was supported by the National Natural Science Foun-

ported by the similarities found in the 5 and 3 terminal UTRs as dation of China (31171818). We thank Nobuhiro Suzuki for fruitful

shown in Fig. S2. discussion and critical reading of the manuscript. We acknowledge

However, FgHV1 differs from CHV1 and CHV2 in the follow- generous gifts of fungal strains from Linghuo Jiang.

ing aspects. Homology search indicated that ORF A of FgHV1 had

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