Molecular characterization of a novel mycovirus in the cultivated mushroom, Lentinula edodes

Abstract

BackgroundIn 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.MethodsA 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.ResultsThe 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.ConclusionsThe results suggest that LeV represents a novel family of mycoviruses, found thus far only among the basidiomycetes.

Molecular characterization of a novel mycovirus from Rhizoctonia fumigata AG-Ba isolate C-314 Baishi

“…RfV1 was clustered with PgLV-1, LeV and RnMBV1 in a separate clade, independent of other genera. Previous studies have indicated that both PgLV-1 and LeV have monopartite dsRNA genomes and their genome organizations are similar to that of RfV1 [24,25,30]. We failed in preliminary attempts to purify virus particles from R. fumigata AG-Ba isolate C-314 Baishi.…”

“…We failed in preliminary attempts to purify virus particles from R. fumigata AG-Ba isolate C-314 Baishi. Attempts to purify the virions of PgLV-1 and LeV were also unsuccessful, suggesting that they may be unencapsidated [24,25]. RnMBV1 consists of two dsRNA segments, dsRNA1 (8931 bp) and dsRNA2 (7180 bp), and is the only member of a newly approved virus family, Megabirnaviridae.…”

“…In RfV1, the sequence AAAUUUC was found at nt 5965-5971 immediately upstream of the stop codon of ORF1, which could act as the shifty heptamer. The -1 frameshift has been demonstrated for viruses with genome organizations similar to that of RfV1, such as the L-A dsRNA virus of Saccharomyces cerevisiae (ScV-L-A) [29], PgLV-1 [25,30], LeV [24] and dsRNA1 of Rosellinia necatrix megabirnavirus 1 (RnMBV1) [31]. The revised ORF2 (nt 5923-9873) encoded 1,315 aa, potentially yielding a 146.72-kDa putative protein (P2) containing the conserved motifs of the RNA-dependent RNA polymerase (RdRp).…”

“…BLAST searches of the NCBI database indicated that P1 shared low but significant amino acid sequence similarity to the putative proteins encoded by ORF1s of two Lentinula edodes mycovirus (LeV) isolates (LeV-HKB and LeV-HKA). P1 contained a NUDIX domain (aa 83-173, pfam00293, NUDIX domain, 8.74e -13 ), which was also present in the putative proteins encoded by the ORF1s of two other mycoviruses, LeV [24] and Phlebiopsis gigantea large virus 1 (PgLV-1) [25]. NUDIX hydrolases are a diverse superfamily of pyrophosphatases that are widespread among eukaryotes, bacteria, archaea and viruses, catalyzing the hydrolysis of nucleoside diphosphates linked to other moieties [26].…”

Extracción y purificación de dsRNAs largos de plantas infectadas con virus y hongos; aplicación en la detección e identificación de virus

“…El ensamble De novo comparando con secuencias de genomas de virus disponibles en la base de datos del NCBI ha permitido identificar nuevas especies de endornavirus y micovirus (Espach et al, 2012;Jo et al, 2015;Khalifa et al, 2016). Utilizando dsRNAs virales, en combinación con secuenciación profunda, los investigadoress han podido obtener secuencias genómicas completas de fitovirus y micovirus (Al Rwahnih et al, 2011;Candresse et al, 2013;Coetzee et al, 2010;Deker y Parker, 2014;Espach et al, 2012;Quito-Avila et al, 2011;Magae, 2012), e identificar elementos que semejan virus en poblaciones microbianas acuáticas (Nerva et al, 2016). La secuenciación profunda de una sola vid reveló un viroma dominado por micovirus (Al Rwahnih et al, 2011).…”