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

Abstract

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 ...

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

“…However, the sequence of the large ORF of MpHV1 was 5= and 3= incomplete. In a separate study, we further characterized SsHV2L, a naturally occurring recombinant strain of Sclerotinia sclerotiorum hypovirus 2 from North America, by determining its complete nucleotide sequence and constructing an infectious clone of the virus (38). Alignments of the predicted amino acid sequences showed that they share from 9 to 29% amino acid sequence identity with previously reported members of the Hypoviridae.…”

“…Infection of individual fungal isolates with the new viruses was confirmed using virus-specific primers in quantitative reverse transcriptase PCR (qRT-PCR) as described previously (38). The origin and host information for fungal isolates shown to be infected with one or more of the identified viruses are presented in Table S2 in the supplemental material.…”

“…A collection of fungal isolates, including, 2 C. truncatum, 48 M. phaseolina, 3 D. longicolla, 84 R. solani, and 138 S. sclerotiorum isolates from various crop plants and locations in North America, maintained by the authors, was used in this research. Cultures were maintained and mycelia were pulverized as described previously (38). Total RNAs were extracted from fungal cultures using the RNeasy minikit (Qiagen, Valentia, CA) and depleted of rRNA with the Ribo-Zero plant kit (Epicentre, Madison, WI).…”

“…The supernatant was recovered and centrifuged at 50,000 rpm in a Beckman Ti71 rotor for 2 h. RNA was extracted from the pellet using the RNeasy minikit. Doublestranded RNAs were extracted from a subset of cultures as previously described (38). A sequencing library was prepared as described above, but without rRNA depletion.…”

Characterization of a Novel Megabirnavirus from Sclerotinia sclerotiorum Reveals Horizontal Gene Transfer from Single-Stranded RNA Virus to Double-Stranded RNA Virus

“…This phenomenon thus reveals that the ORFA-encoded protein of L2-dsRNA/SsMBV1 is produced as a consequence of recombinational events between different viruses. Moreover, several S. sclerotiorum isolates have been confirmed to be infected with hypoviruses (23,61,62,63), suggesting that some hosts provided opportunities for HGT between hypovirus and SsMBV1 during evolutionary processes. Based on the data presented here, we hypothesize that HGT might have occurred from a hypovirus to a dsRNA genomic component of SsMBV1 (or alternatively to an unusually large satellite-like dsRNA dependent on SsMBV1 for replication and encapsidation).…”