Short interspersed nuclear element (SINE) sequences in the genome of the human pathogenic fungus Aspergillus fumigatus Af293.

Copyright: © 2016 The Authors. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Kanhayuwa L, Coutts RHA (2016) Short Interspersed Nuclear Element (SINE) Sequences in the Genome of the Human Pathogenic Fungus Aspergillus fumigatus Af293. PLoS ONE 11(10): e0163215. https://doi.org/10.1371/journal.pone.0163215.Novel families of short interspersed nuclear element (SINE) sequences in the human pathogenic fungus Aspergillus fumigatus, clinical isolate Af293, were identified and categorised into tRNA-related and 5S rRNA-related SINEs. Eight predicted tRNA-related SINE families originating from different tRNAs, and nominated as AfuSINE2 sequences, contained target site duplications of short direct repeat sequences (4-14 bp) flanking the elements, an extended tRNA-unrelated region and typical features of RNA polymerase III promoter sequences. The elements ranged in size from 140-493 bp and were present in low copy number in the genome and five out of eight were actively transcribed. One putative tRNAArg-derived sequence, AfuSINE2-1a possessed a unique feature of repeated trinucleotide ACT residues at its 3'-terminus. This element was similar in sequence to the I-4_AO element found in A. oryzae and an I-1_AF long nuclear interspersed element-like sequence identified in A. fumigatus Af293. Families of 5S rRNA-related SINE sequences, nominated as AfuSINE3, were also identified and their 5'-5S rRNA-related regions show 50-65% and 60-75% similarity to respectively A. fumigatus 5S rRNAs and SINE3-1_AO found in A. oryzae. A. fumigatus Af293 contains five copies of AfuSINE3 sequences ranging in size from 259-343 bp and two out of five AfuSINE3 sequences were actively transcribed. Investigations on AfuSINE distribution in the fungal genome revealed that the elements are enriched in pericentromeric and subtelomeric regions and inserted within gene-rich regions. We also demonstrated that some, but not all, AfuSINE sequences are targeted by host RNA silencing mechanisms. Finally, we demonstrated that infection of the fungus with mycoviruses had no apparent effects on SINE activity.Peer reviewedFinal Published versio

Development of silencing vectors for Aspergillus fumigatus based on mycoviruses and short interspersed nuclear elements

A novel mycovirus named Aspergillus fumigatus tetramycovirus-1 (AfuTmV-1) was discovered and characterized in the human pathogenic fungus, A. fumigatus clinical isolate Af293. The virus reveals several unique features not previously found in double-stranded RNA (dsRNA) viruses and represents the first dsRNA that is infectious both as a purified entity and a naked dsRNA. The AfuTmV-1 is an unencapsidated dsRNA mycovirus comprised of four genomic segments, ranging in size from 1.1 to 2.4 kbp. The largest component encodes a putative viral RNA-dependent RNA polymerase (RdRP) where the sequence of the most highly conserved motif changes from GDDX to GDNQ. The third largest dsRNA encodes an S-adenosyl methionine-dependent methyltransferase (SAM) capping enzyme and the smallest dsRNA encodes a proline-alanine rich protein. Short interspersed nuclear elements (SINEs) were also identified in the fungal genome. Identification of the elements revealed tRNA-related and 5S rRNA-related SINE families which showed variation in transcription activity and copy number. AfuTmV-1 sequences together with SINEs were subsequently exploited to develop alternative tools for silencing genes in A. fumigatus. A truncated AfuTmV-1 based vector was successfully constructed and used as a prototype vector for generating a recombinant virus-induced gene silencing (VIGS) vector. Transcriptional fusion SINE-derived vectors were also developed to silence an ALB1/PKSP gene responsible for conidial pigmentation. With anticipation that the development will provide a powerful reverse genetic tool for functional genomics studies to identify key elements involved in fungal pathogenicity and also provide a medical benefit in exploiting mycoviruses as a future therapeutic agent against fungal infections.Open Acces

Detection of small RNA molecules homologous to <i>AfuSINE</i> sequences.

<p>Total RNAs isolated with Trizol were electrophoresed on 1.5% agarose gels (a). After separation by PEG precipitation, high molecular weight RNAs (HMW RNAs) and low molecular weight RNAs (LMW RNAs) were electrophoresed on 1.5% agarose gels (b). The LMW fractions from 400 ng/μl of total RNA were resolved on 15% (w/v) polyacrylamide Tris-borate-EDTA-urea gels and stained with SYBR Gold Nucleic Acid Gel Stain (c). Northern blot hybridization of small RNAs homologous to <i>AfuSINE</i> sequences in <i>A</i>. <i>fumigatus</i> Af293 (d). LMW RNA fractions were isolated using TRIzol and 5 μg RNA sample was loaded into each well for northern blot analysis. Only small RNAs (<40 nt; arrowed) from the antisense strand were detected.</p

Structure of <i>LINE</i>-like element.

<p>Structure of <i>LINE</i>-like element (LLE#4_3.0; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163215#pone.0163215.ref022" target="_blank">22</a>]) on <i>A</i>. <i>fumigatus</i> Af293 chromosome 4 showing insertion of the <i>AfuSINE2_4c</i> sequence next to the LLE RT.</p

Mapping of <i>SINE</i>-like sequences of the <i>A</i>. <i>fumigatus</i> Af293 genome.

<p>Mapping of <i>SINE</i>-like sequences on eight chromosomes of the <i>A</i>. <i>fumigatus</i> Af293 genome including previously described <i>LINE</i>-like sequences (LLEs; [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163215#pone.0163215.ref022" target="_blank">22</a>]). Chromosome numbers are shown on the left and the size of each chromosome is shown on the right.</p

Structure of the <i>AfuSINE2-1a</i> tRNA-derived <i>SINE</i>.

<p>Structure of the <i>AfuSINE2-1a</i> tRNA-derived <i>SINE</i> found in <i>A</i>. <i>fumigatus</i> Af293 (a) and the alignment of its 3'-terminus which is related to <i>I-4_AO LINE</i> (b).</p

Consensus A and B box promoter sequences of RNA pol III in tRNA-derived <i>AfuSINE</i> sequences (<i>AfuSINE2s</i>).

<p>Consensus A and B box promoter sequences of RNA pol III in tRNA-derived <i>AfuSINE</i> sequences (<i>AfuSINE2s</i>).</p

Sequence determination of a quadripartite dsRNA virus isolated from Aspergillus foetidus

Virus infection of Aspergillus foetidus was documented over 40 years ago and was one of the first mycovirus infections described in a filamentous fungus. The virus, named Aspergillus foetidus virus (AfV), contains at least two types of icosahedral particles, called AfV-fast (-F) and AfV-slow (-S) virions, based on their relative electrophoretic mobilities. Here, we report the complete nucleotide sequence of the AfV-F genome isolated from virions purified from the prototype isolate of the fungus. The AfV-F double-stranded (ds) RNA genome is tetra-segmented, and the plus strands of each of the four segments, but not the minus strands, are polyadenylated. The organisation and sequences of the four AfV-F dsRNAs are similar to those described for Alternaria alternata virus 1, which we propose is a member of an emerging mycovirus genus ("Alternavirus") and family ("Alternaviridae"), which also includes AfV-F.Peer reviewe