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Deep sequencing of mycovirus-derived small RNAs from Botrytis species.

ABSTRACT: RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, although the analysis of public genome databases identified two Dicer-like genes in B. cinerea, as in most of the ascomycetes sequenced to date. In this work, we used deep sequencing to study the virus-derived small RNA (vsiRNA) populations from different mycoviruses infecting field isolates of Botrytis spp. The mycoviruses under study belong to different genera and species, and have different types of genome [double-stranded RNA (dsRNA), (+)single-stranded RNA (ssRNA) and (-)ssRNA]. In general, vsiRNAs derived from mycoviruses are mostly of 21, 20 and 22 nucleotides in length, possess sense or antisense orientation, either in a similar ratio or with a predominance of sense polarity depending on the virus species, have predominantly U at their 5' end, and are unevenly distributed along the viral genome, showing conspicuous hotspots of vsiRNA accumulation. These characteristics reveal striking similarities with vsiRNAs produced by plant viruses, suggesting similar pathways of viral targeting in plants and fungi. We have shown that the fungal RNA silencing machinery acts against the mycoviruses used in this work in a similar manner independent of their viral or fungal origin.

SUBMITTER: Donaire L

PROVIDER: S-EPMC6638239 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Deep sequencing of mycovirus-derived small RNAs from Botrytis species.

Donaire Livia L Ayllón María A MA

Molecular plant pathology 20161012 8

RNA silencing is an ancient regulatory mechanism operating in all eukaryotic cells. In fungi, it was first discovered in Neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in Cryphonectria parasitica and, later, in several fungal species. There is little evidence of the antiviral potential of RNA silencing in the phytopathogenic species of the fungal genus Botrytis. Moreover, little is known about the RNA silencing components in these fungi, a ...[more]

PMID: 27578449

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Project description:BackgroundSmall RNA (sRNA)-guided RNA silencing is a critical antiviral defense mechanism employed by a variety of eukaryotic organisms. Although the induction of RNA silencing by bipartite and monopartite begomoviruses has been described in plants, the nature of begomovirus/betasatellite complexes remains undefined.Methodology/principal findingsSolanum lycopersicum plant leaves systemically infected with Tomato yellow leaf curl China virus (TYLCCNV) alone or together with its associated betasatellite (TYLCCNB), and Nicotiana benthamiana plant leaves systemically infected with TYLCCNV alone, or together with TYLCCNB or with mutant TYLCCNB were harvested for RNA extraction; sRNA cDNA libraries were then constructed and submitted to Solexa-based deep sequencing. Both sense and anti-sense TYLCCNV and TYLCCNB-derived sRNAs (V-sRNAs and S-sRNAs) accumulated preferentially as 22 nucleotide species in infected S. lycopersicum and N. benthamiana plants. High resolution mapping of V-sRNAs and S-sRNAs revealed heterogeneous distribution of V-sRNA and S-sRNA sequences across the TYLCCNV and TYLCCNB genomes. In TYLCCNV-infected S. lycopersicum or N. benthamiana and TYLCCNV and βC1-mutant TYLCCNB co-infected N. benthamiana plants, the primary TYLCCNV targets were AV2 and the 5' terminus of AV1. In TYLCCNV and betasatellite-infected plants, the number of V-sRNAs targeting this region decreased and the production of V-sRNAs increased corresponding to the overlapping regions of AC2 and AC3, as well as the 3' terminal of AC1. βC1 is the primary determinant mediating symptom induction and also the primary silencing target of the TYLCCNB genome even in its mutated form.Conclusions/significanceWe report the first high-resolution sRNA map for a monopartite begomovirus and its associated betasatellite using Solexa-based deep sequencing. Our results suggest that viral transcript might act as RDR substrates resulting in dsRNA and secondary siRNA production. In addition, the betasatellite affected the amount of V-sRNAs detected in S. lycopersicum and N. benthamiana plants.

Project description:Sclerotinia sclerotiorum, an important phytopathogenic fungus, harbors rich diversity of mycoviruses. Lately, more mycoviruses can be successfully and accurately discovered by deep sequencing, especially those that could not be detected by traditional double-stranded RNA (dsRNA) extraction. Previously, we reported that the hypovirulent S. sclerotiorum strain SZ-150 is coinfected by Sclerotinia sclerotiorum hypovirus 1 (SsHV1) and its related satellite RNA. Here, aside from SsHV1, we detected two other mycoviruses, Sclerotinia sclerotiorum botybirnavirus 3 (SsBV3/SZ-150) and Sclerotinia sclerotiorum mycotymovirus 1 (SsMTV1/SZ-150), coinfecting strain SZ-150, by deep sequencing and assembly of mycovirus-derived small RNAs and determined their full-length genomes. The genome of SsBV3/SZ-150 was found to be composed of two linear dsRNA segments, 6,212, and 5,880 bp in size, respectively. Each dsRNA segment of SsBV3/SZ-150 contains a large open reading frame (ORF) encoding RNA-dependent RNA polymerase (RdRp) and a hypothetical protein. The whole genome of SsBV3/SZ-150 shares more than 95% sequence identity with Botrytis porri botybirnavirus 1 (BpBV1) at the nucleotide (nt) or amino acid level. Thus, SsBV3/SZ-150 was assumed to be a strain of BpBV1. The genome of SsMTV1/SZ-150 consists of 6,391 nt excluding the poly(A) tail. SsMTV1/SZ-150 was predicted to contain a large ORF that encodes a putative replication-associated polyprotein (RP) with three conserved domains of viral RNA methyltransferase, viral RNA helicase, and RdRp. Phylogenetic analyses suggest that SsMTV1/SZ-150 is related, albeit distantly, to members of the family Tymoviridae. Analysis of the small RNAs derived from SsBV3/SZ-150 and SsMTV1/SZ-150 revealed that small-RNA lengths mainly range from 20 to 24 nt, with a peak at 22 nt, and the most abundant 5'-terminal nucleotide is uridine, suggesting that the Dicer 2 and Argonaute 1, two key components in the RNA inference pathway, may play important roles in the resistance to mycoviral infection in S. sclerotiorum. Neither SsBV3/SZ-150 nor SsMTV1/SZ-150 is a causal agent of hypovirulence in strain SZ-150.

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Dataset Information

Deep sequencing of mycovirus-derived small RNAs from Botrytis species.

Publications

Deep sequencing of mycovirus-derived small RNAs from Botrytis species.

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