Molecular characterization of a novel mycovirus in the cultivated mushroom, Lentinula edodes
© Magae; licensee BioMed Central Ltd. 2012
Received: 1 June 2011
Accepted: 6 March 2012
Published: 6 March 2012
In the 1970s, mycoviruses were identified that infected the edible mushroom Lentinula edodes (shiitake), but they were not regarded as causal agents for mushroom diseases. None of their genes has been sequenced. In this study, the dsRNA genome of a mycovirus recently found in a shiitake commercial strain was sequenced and its molecular structure was characterized.
A cDNA library was constructed from a dsRNA purified from the fruiting body of L. edodes. The virus was tentatively named L. edodes mycovirus HKB (LeV). Based on the deduced RNA-dependent RNA polymerase (RdRp) sequence, phylogenetic analysis of LeV was conducted. Because no virion particles associated with the dsRNA were observed by electron microscopic observation, atomic force microscopy (AFM) observation was chosen for achieving molecular imaging of the virus.
The 11,282-bp genome of LeV was obtained. The genome encoded two open reading frames (ORFs). ORF1 coded for a hypothetical protein and ORF2 for a putative RdRp, respectively. In addition, a region coding for a NUDIX domain was present in ORF1. There was a 62-bp intergenic region between ORF1 and RdRp. Similarity with coat protein of mycoviruses was not found within the whole sequence. Based on phylogenetic analysis of the putative RdRp sequence, LeV grouped into a clade with dsRNA found in the basidiomycetes Phlebiopsis gigantea and Helicobasidium mompa. The clade was placed apart from the Totiviridae and Chrysoviridae families. As suggested from the genome sequence, AFM revealed that the structure of LeV was linear unencapsidated dsRNA.
The results suggest that LeV represents a novel family of mycoviruses, found thus far only among the basidiomycetes.
In the 1970s, viruses that infect the cultivated mushroom Lentinula edodes, or shiitake, were extensively studied in Japan [1–3], and three morphologically distinct viruses were detected by electron microscopy [1, 3]. However, unlike La France disease of the white button mushroom [4, 5], mycoviruses have not been associated with shiitake diseases because these mycoviruses have commonly been found in healthy fruiting bodies [1, 3]. In the USA, dsRNAs have also been observed in shiitake strains, but these appeared to be latent . In the 1970s, shiitake cultivation was performed by inoculating mycelium spawn on oak logs; however, this labor-intensive method was gradually replaced by indoor cultivation using sawdust substrate supplemented with rice bran. Currently in Japan, about 75,016 t (82% of all shiitake) are produced indoors annually using bag cultures with a sawdust-based substrate . In a bag culture, the shiitake mycelium is fully grown in the substrate until brown pigment is produced outside and the substrate becomes stiff. Complete browning of the exterior surface of the substrate is an important marker that normal fruiting bodies will develop in the following stage of cultivation. However, abnormal symptoms are occasionally observed in bag cultures, such as the growth of white or fluffy mycelia on the surface of substrate, inadequate or imperfect substrate browning , and malformations of the fruiting body. These symptoms often result in serious economic losses. Whether or not these abnormalities are associated with mycovirus is unknown. As a first step toward addressing that question, 46 shiitake isolates belonging to 11 commercial strains were examined for mycovirus infections . As a result, dsRNA was found in two isolates; one showed imperfect browning and the other was asymptomatic. Agarose gel analysis showed that the isolate with imperfect browning contained several dsRNAs, but the asymptomatic isolate contained only a single dsRNA. In this study, the single dsRNA was tentatively designated as Lentinula edodes mycovirus HKB (LeV) and was sequenced.
Three fruiting bodies were disrupted in 60 ml of 0.1 M phosphate buffer, and the virus fraction was precipitated with 10% PEG 8000 and 0.15 M NaCl, as described previously . The PEG precipitate was suspended in TES (10 mM Tris-HCl, 1 mM EDTA, 0.15 M NaCl, pH 7.0), and total RNA was isolated using the QIAmp Viral RNA Mini Kit (Qiagen) according to the manufacturer's instructions. Then dsRNA was isolated from the viral RNA by DNase I (Promega) digestion for 30 min at 37°C, followed by S1 nuclease (TaKaRa) digestion. The resulting dsRNA was concentrated in nuclease-free water by filtration through Ultrafree 0.5 100 K centrifugal filters (Millipore) several times to remove degraded nucleic acids and salts. Finally, dsRNA was purified with the RNeasy Mini Elute Cleanup Kit (Qiagen).
cDNA library construction and sequencing of dsRNA
The purified dsRNA served as a template for cDNA synthesis by random priming with the PrimeScript 1st strand cDNA Synthesis Kit (TaKaRa) according to the standard protocol, except that the denaturing condition was changed from 65°C, 5 min to 98°C, 1 min. The resulting cDNAs were electrophoresed in an agarose gel, and cDNAs sized 1.5-2.0 kb were extracted. The cDNAs were blunt-ended with T4 DNA polymerase, ligated into pUC118/HincII/BAP (TaKaRa), and transformed into E. coli DH10B cells by electroporation (Gene Pulser, Bio-Rad). The resulting cloned DNA was sequenced with the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) with M13 forward and reverse primers. A contiguous region (contig) was assembled with Sequencher™ 4.6 (Gene Codes Corp).
AFM microscopic observation
The PEG precipitate was suspended in 500 μl of TE and filtered through a 0.2-μm filter (Millipore), and 1 μl of the filtrate was diluted with 50 μl TE + 10 mM MgCl2. The sample was observed using atomic force microscopy (AFM) as previously described . A total of 10 μl of the RNA solution was dropped onto freshly cleaved muscovite mica (1 × 1 cm), which after standing for several minutes was washed with distilled water. The sample was dried under a stream of nitrogen. Observations were performed on a MFP-3D (Asylum Research) in the tapping mode in air, using a silicon cantilever OMCL-AC240TS (Olympus). Fields of 2 μm × 2 μm were scanned at a frequency of < 2 Hz. To confirm that the AFM image observed was RNA, AFM imaging was also performed with PEG precipitate after RNaseA digestion. The length of the RNA molecule was measured using software developed by the Research Institute of Biomolecule Metrology Co., Ltd. (Japanese patent P2000-230823A).
RdRp sequences of viruses included in the phylogenetic tree
Lentinula edodes mycovirus HKB LeVHKB
Phlebiopsis gigantea mycovirus dsRNA1 PgV1
Helicobasidium mompa V670 L2-dsRNA virus HmV670
Helminthosporium victoriae 145S virus HvV145S
Verticillium chrysogenum virus VcV
Cryphonectria nitschkei chrysovirus 1 CnCV1
Penicillium chrysogenum virus PcV
Anthurium mosaic-associated virus AmV
Grapevine associated chrysovirus-1 GaCV1
Aspergillus fumigatus chrysovirus AfCV
Circulifer tenellus virus 1 CiTV1
Spissistilus festinus virus 1 SpFV1
Saccharomyces cerevisiae virus L-A ScVLA
Sphaeropsis sapinea RNA virus 1 SsRV1
Results and discussion
AFM microscopic observation
Hypothesis of unencapsidated virus in the basidiomycetes
L. edodes is a white-rot basidiomycete and P. gigantea is also a basidiomycete that causes white rot in conifer logs and stumps [32, 33]. Often, P. gigantea is isolated from bark beetle, as is Lentinula boryana, a fungus belonging to the same family as L. edodes . Virion structure is necessary for a virus to infect and exit from the host cell and gain a greater probability of propagation. Because bark beetles feed on basidiomycete fungi , the evolution of unencapsidated virus in white-rot basidiomycetes may have been achieved through their association with wood-feeding insects. Because their host can be efficiently transferred to new environments by bark beetles, the coat protein would no longer be necessary for virus propagation. Additionally, the conserved amino acids of putative RdRp molecules of unencapsidated viruses isolated from plant-feeding insects, such as the alfalfa hopper and beet leafhopper , are significantly similar to LeV (Figure 4) (Table 1). This fact also supports the hypothesis that unencapsidated species of mycovirus present in the basidiomycetes might be closely associated with insects.
dsRNA found in a commercial strain of Lentinula edodes (shiitake) (designated as L. edodes mycovirus HKB; LeV) was sequenced. The 11.8-kb genome contained two ORFs. ORF1 coded for a hypothetical protein containing a NUDIX domain. Previously, viral NUDIX domain was found only in Poxviruses. LeV is the only mycovirus that codes NUDIX domain. ORF2 coded for RdRp with high similarity with Totivirus and Chrysovirus. The genome did not code for a coat protein, and AFM observation revealed LeV to be unencapsidated linear dsRNA. The results show that LeV is a novel mycovirus with a monopartite dsRNA genome that is closely related to Totivirus, but that it does not form a virion particle and might represent a new class of mycovirus.
I would like to thank China Ohta, Katsumasa Eda, and Sumio Ayusawa of Hokken Co., Ltd. for providing fruiting bodies of strain HKB and all the collaboration during the project granted by the Agriculture, Forestry and Fisheries Research Council of Japan. I would also like to thank Dr. Junji Magae for valuable suggestions. Construction of cDNA libraries and sequencing was performed by TaKaRa Bio Inc. AFM was performed at the Research Institute of Biomolecule Metrology Co., Ltd., (RIBM) Enokido, Tsukuba, Ibaraki, 305-0853, Japan.
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