Novel Hypovirulence-Associated RNA Mycovirus in the Plant-Pathogenic Fungus Botrytis cinerea: Molecular and Biological Characterization

Abstract

Botrytis cinerea is a pathogenic fungus causing gray mold on numerous economically important crops and ornamental plants. This study was conducted to characterize the biological and molecular features of a novel RNA mycovirus, Botrytis cinerea RNA virus 1 (BcRV1), in the hypovirulent strain BerBc-1 of B. cinerea. The genome of BcRV1 is 8,952 bp long with two putative overlapped open reading frames (ORFs), ORF1 and ORF2, coding for a hypothetical polypeptide (P1) and RNA-dependent RNA polymerase (RdRp), respectively. A ؊1 frameshifting region (designated the KNOT element) containing a shifty heptamer, a heptanucleotide spacer, and an H-type pseudoknot was predicted in the junction region of ORF1 and ORF2. The ؊1 frameshifting role of the KNOT element was experimentally confirmed through determination of the production of the fusion protein red fluorescent protein (RFP)-green fluorescent protein (GFP) by the plasmid containing the construct dsRed-KNOT-eGFP in Escherichia coli. BcRV1 belongs to a taxonomically unassigned double-stranded RNA (dsRNA) mycovirus group. It is closely related to grapevine-associated totivirus 2 and Sclerotinia sclerotiorum nonsegmented virus L. BcRV1 in strain BerBc-1 was found capable of being transmitted vertically through macroconidia and horizontally to other B. cinerea strains through hyphal contact. The presence of BcRV1 was found to be positively correlated with hypovirulence in B. cinerea, with the attenuation effects of BcRV1 on mycelial growth and pathogenicity being greatly affected by the accumulation level of BcRV1. M ycoviruses, or fungal viruses, are viruses infecting filamentous fungi and yeasts (1, 2). Most mycoviruses reported so far are either positive-single-stranded RNA (ϩssRNA) viruses, including mycoviruses in the families Hypoviridae and Narnaviridae, or double-stranded RNA (dsRNA) viruses, including mycoviruses in the families Chrysoviridae, Megabirnaviridae, Partitiviridae, Reoviridae, and Totiviridae (1, 3). Recently, mycoviruses with negative-single-stranded RNA genomes (ϪssRNA) were reported (4, 5). Moreover, Yu et al. (6) reported a single-stranded DNA (ssDNA) mycovirus, namely, Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1), in Sclerotinia sclerotiorum, the causal agent of Sclerotinia stem rot of oilseed rape (Brassica napus).Mycoviruses are widespread in all major taxonomic groups of fungi, including many plant pathogens (2). In most cases, mycovirus infection appears symptomless on the host fungi, usually called a latent or cryptic infection (1). However, infection by some mycoviruses in the families Hypoviridae, Megabirnaviridae, Narnaviridae, Partitiviridae, and Reoviridae or by the unassigned ϪssRNA and ssDNA mycoviruses can cause visible abnormal symptoms on the host fungi, including reduced mycelial growth, reduced production of spores and/or sclerotia, suppressed biosynthesis of secondary metabolites, and attenuated aggressiveness or virulence (2, 3, 5-7). Some hypovirulence-causing mycoviruses have been reported to be prom...

Identification of Diverse Mycoviruses through Metatranscriptomics Characterization of the Viromes of Five Major Fungal Plant Pathogens

“…Like FgV3, the MpDSRV2 sequence was about 9.1 kb and contained two large ORFs, suggesting that the assembled sequence represents a nearly complete virus genome. The predicted amino acid sequence of ORF1 was similar to the products of the first ORFs of other unclassified bicistroinc dsRNA mycoviruses with genomes of 8.9 to 9.5 kb (66)(67)(68)(69). Unlike the genomes of the other large bicistronic dsRNA mycoviruses, the two ORFs in MpDSRV2 overlapped by 28 nt, and ORF2 could be translated by a Ϫ1 frameshift from ORF1.…”

Short communication: Effect of mycoviruses on growth, spore germination and pathogenicity of the fungus Fusarium circinatum

“…A reduction in the mycelial growth of different pathogens infested with mycoviruses has been reported to be related to hypovirulence in fungi in several studies (Bottacin et al, 1994;Ahn & Lee, 2001;Castro et al, 2003;Robin et al, 2010;Rodríguez-García et al, 2014;Zheng et al, 2014;Yu et al, 2015). Nevertheless, the effect on mycelial growth may also be determined by the species (Vainio et al, 2012;Hyder et al, 2013), isolate (Hunst et al, 1986;Rodríguez-García et al, 2014;Yu et al, 2015) temperature (Vainio et al, 2010;Bryner & Rigling, 2011;Romeralo et al, 2012;Zamora et al, 2016) or the type of mycovirus (Cryphonectria parasitica CHV1, mycovirus hypovirulences). These factors, among others, may explain the inconsistencies in the findings of different studies.…”

The dsRNA Virus Papaya Meleira Virus and an ssRNA Virus Are Associated with Papaya Sticky Disease

“…The same putative frameshifting sequence is present in PMeV-RN, and similar structures have been identified in the five viruses most closely related to PMeV (PgV-2, FgV-3, PiRV-3, SsNsV-L and BcRV-1). In these viruses, the slippery sequences differ from the general heptanucleotide XXXYYYZ, where X represents any nucleotide, Y represents A or U, and Z represents A, C or U [ 22 , 24 , 25 ]. Following the slippery sequence there is a stable secondary structure, such as a pseudoknot or hairpin [ 26 ].…”