Identification of novel double-stranded RNA mycoviruses of Fusarium virguliforme and evidence of their effects on virulence

Marvelli, Rita A.; Hobbs, H. A.; Li, Shuxian; McCoppin, Nancy K.; Domier, Leslie L.; Hartman, G. L.; Eastburn, Darin M.

doi:10.1007/s00705-013-1760-1

Cited by 32 publications

(38 citation statements)

References 21 publications

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“…The sequence of BcRV1 P1 has three motifs (I to III) (Fig. 3) similar to those found in the P1 homologs in seven other dsRNA viruses, including grapevine-associated totivirus 2 (GaTV2) (41), Fusarium graminearum dsRNA mycovirus 3 (FgV3) (42), Fusarium virguliforme dsRNA mycovirus 1 (FvV1) (43), Fusarium virguliforme dsRNA mycovirus 2 (FvV2) (43), Phlebiopsis gigantea mycovirus dsRNA 2 (PgV2) (44), Phytophthora infestans RNA virus 3 (PiRV3) (45), and Sclerotinia sclerotiorum nonsegmented virus L (SsNsV-L) (46). The identity of BcRV1 P1 to the P1 homologs in SsNsV-L (77%) and GaTV2 (93%) is high, whereas to the P1 homologs in the remaining five dsRNA viruses it is low (22 to 45%) (Table 1).…”

Section: Resultsmentioning

confidence: 63%

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

Sang

et al. 2015

Appl Environ Microbiol

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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...

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Section: Resultsmentioning

confidence: 63%

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

Sang

et al. 2015

Appl Environ Microbiol

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“…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.…”

Section: Resultsmentioning

confidence: 81%

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

Marzano

Nelson

Ajayi-Oyetunde

et al. 2016

J Virol

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225

224

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Mycoviruses can have a marked effect on natural fungal communities and influence plant health and productivity. However, a comprehensive picture of mycoviral diversity is still lacking. To characterize the viromes of five widely dispersed plant-pathogenic fungi, Colletotrichum truncatum, Macrophomina phaseolina, Diaporthe longicolla, Rhizoctonia solani, and Sclerotinia sclerotiorum, a high-throughput sequencing-based metatranscriptomic approach was used to detect viral sequences. Total RNA and double-stranded RNA (dsRNA) from mycelia and RNA from samples enriched for virus particles were sequenced. Sequence data were assembled de novo, and contigs with predicted amino acid sequence similarities to viruses in the nonredundant protein database were selected. The analysis identified 72 partial or complete genome segments representing 66 previously undescribed mycoviruses. Using primers specific for each viral contig, at least one fungal isolate was identified that contained each virus. The novel mycoviruses showed affinity with 15 distinct lineages: Barnaviridae, Benyviridae, Chrysoviridae, Endornaviridae, Fusariviridae, Hypoviridae, Mononegavirales, Narnaviridae, Ophioviridae, Ourmiavirus, Partitiviridae, Tombusviridae, Totiviridae, Tymoviridae, and Virgaviridae. More than half of the viral sequences were predicted to be members of the Mitovirus genus in the family Narnaviridae, which replicate within mitochondria. Five viral sequences showed strong affinity with three families (Benyviridae, Ophioviridae, and Virgaviridae) that previously contained no mycovirus species. The genomic information provides insight into the diversity and taxonomy of mycoviruses and coevolution of mycoviruses and their fungal hosts. IMPORTANCEPlant-pathogenic fungi reduce crop yields, which affects food security worldwide. Plant host resistance is considered a sustainable disease management option but may often be incomplete or lacking for some crops to certain fungal pathogens or strains. In addition, the rising issues of fungicide resistance demand alternative strategies to reduce the negative impacts of fungal pathogens. Those fungus-infecting viruses (mycoviruses) that attenuate fungal virulence may be welcome additions for mitigation of plant diseases. By high-throughput sequencing of the RNAs from 275 isolates of five fungal plant pathogens, 66 previously undescribed mycoviruses were identified. In addition to identifying new potential biological control agents, these results expand the grand view of the diversity of mycoviruses and provide possible insights into the importance of intracellular and extracellular transmission in fungus-virus coevolution. R ecent metatranscriptomic and metagenomic studies of animals, fungi, insects, plants, and environmental samples have shown that mycoviruses are ubiquitous in nature (1-10). Analyses of viral metagenomes (i.e., viromes) of environmental samples suggest that the field of virology has discovered less than 1% of the existing viral diversity, and the rate of discovery by metage...

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“…High-throughput sequencing technologies for metagenomics have opened a new era for discovery of viruses that are relatively undersurveyed in nature (51-54), including mycoviruses (55). In this study, bioinformatic analysis of HTS data was used to assemble a nearly complete genome sequence of a recombined isolate of SsHV2 and SsEV1 without first purifying dsRNA.…”

Section: Discussionmentioning

confidence: 99%

Transfection of Sclerotinia sclerotiorum with In Vitro Transcripts of a Naturally Occurring Interspecific Recombinant of Sclerotinia sclerotiorum Hypovirus 2 Significantly Reduces Virulence of the Fungus

Marzano

Hobbs

Nelson

et al. 2015

J Virol

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A recombinant strain of Sclerotinia sclerotiorum hypovirus 2 (SsHV2) was identified from a North American Sclerotinia sclerotiorum isolate (328) from lettuce (Lactuca sativa L.) by high-throughput sequencing of total RNA. The 5=-and 3=-terminal regions of the genome were determined by rapid amplification of cDNA ends. The assembled nucleotide sequence was up to 92% identical to two recently reported SsHV2 strains but contained a deletion near its 5= terminus of more than 1.2 kb relative to the other SsHV2 strains and an insertion of 524 nucleotides (nt) that was distantly related to Valsa ceratosperma hypovirus 1. This suggests that the new isolate is a heterologous recombinant of SsHV2 with a yet-uncharacterized hypovirus. We named the new strain Sclerotinia sclerotiorum hypovirus 2 Lactuca (SsHV2L) and deposited the sequence in GenBank with accession number KF898354. Sclerotinia sclerotiorum isolate 328 was coinfected with a strain of Sclerotinia sclerotiorum endornavirus 1 and was debilitated compared to cultures of the same isolate that had been cured of virus infection by cycloheximide treatment and hyphal tipping. To determine whether SsHV2L alone could induce hypovirulence in S. sclerotiorum, a full-length cDNA of the 14,538-nt viral genome was cloned. Transcripts corresponding to the viral RNA were synthesized in vitro and transfected into a virus-free isolate of S. sclerotiorum, DK3. Isolate DK3 transfected with SsHV2L was hypovirulent on soybean and lettuce and exhibited delayed maturation of sclerotia relative to virus-free DK3, completing Koch's postulates for the association of hypovirulence with SsHV2L. IMPORTANCEA cosmopolitan fungus, Sclerotinia sclerotiorum infects more than 400 plant species and causes a plant disease known as white mold that produces significant yield losses in major crops annually. Mycoviruses have been used successfully to reduce losses caused by fungal plant pathogens, but definitive relationships between hypovirus infections and hypovirulence in S. sclerotiorum were lacking. By establishing a cause-and-effect relationship between Sclerotinia sclerotiorum hypovirus Lactuca (SsHV2L) infection and the reduction in host virulence, we showed direct evidence that hypoviruses have the potential to reduce the severity of white mold disease. In addition to intraspecific recombination, this study showed that recent interspecific recombination is an important factor shaping viral genomes. The construction of an infectious clone of SsHV2L allows future exploration of the interactions between SsHV2L and S. sclerotiorum, a widespread fungal pathogen of plants. Sclerotinia sclerotiorum (Lib.) de Bary is a cosmopolitan fungal plant pathogen that causes necrotic diseases (e.g., Sclerotinia stem rot) in more than 400 plant species, which result in yield losses in major crops each year (1-3). However, the diseases caused by S. sclerotiorum have not been adequately controlled by conventional technologies thus far (4, 5). A number of mycoviruses have been molecularly characterized and ...

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Identification of novel double-stranded RNA mycoviruses of Fusarium virguliforme and evidence of their effects on virulence

Cited by 32 publications

References 21 publications

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

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

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

Transfection of Sclerotinia sclerotiorum with In Vitro Transcripts of a Naturally Occurring Interspecific Recombinant of Sclerotinia sclerotiorum Hypovirus 2 Significantly Reduces Virulence of the Fungus

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