High-throughput sequencing reveals small RNAs involved in ASGV infection
© Visser et al.; licensee BioMed Central Ltd. 2014
Received: 30 April 2014
Accepted: 26 June 2014
Published: 7 July 2014
Plant small RNAs (sRNAs) associated with virulent virus infections have been reported by previous studies, while the involvement of sRNAs in latent virus infection remains largely uncharacterised. Apple trees show a high degree of resistance and tolerance to viral infections. We analysed two sRNA deep sequencing datasets, prepared from different RNA size fractions, to identify sRNAs involved in Apple stem grooving virus (ASGV) infection.
sRNA analysis revealed virus-derived siRNAs (vsiRNAs) originating from two ASGV genetic variants. A vsiRNA profile for one of the ASGV variants was also generated showing an increase in siRNA production towards the 3′ end of the virus genome. Virus-derived sRNAs longer than those previously analysed were also observed in the sequencing data. Additionally, tRNA-derived sRNAs were identified and characterised. These sRNAs covered a broad size-range and originated from both ends of the mature tRNAs as well as from their central regions. Several tRNA-derived sRNAs showed differential regulation due to ASGV infection. No changes in microRNA, natural-antisense transcript siRNA, phased-siRNA and repeat-associated siRNA levels were observed.
This study is the first report on the apple sRNA-response to virus infection. The results revealed the vsiRNAs profile of an ASGV variant, as well as the alteration of the tRNA-derived sRNA profile in response to latent virus infection. It also highlights the importance of library preparation in the interpretation of high-throughput sequencing data.
KeywordsApple stem grooving virus Next-generation sequencing Plant-virus interaction tRNA-derived fragment tRNA-half Virus-derived small interfering RNA
The domesticated apple, Malus x domestica (M. x domestica), has a wide range of infectious agents, which include fungi, bacteria, phytoplasma, viruses and viroids. One such virus, Apple stem grooving virus (ASGV), is the type member of the genus Capillovirus (family Flexiviridae) . It is a positive-sense RNA virus with a genome of approximately 6.5 kb, which is organised into two overlapping open reading frames (ORFs) . ASGV infection is mostly symptomless (latent) in apple cultivars, depending on the virus strain, however some cultivars are susceptible and may develop severe symptoms such as xylem pitting and grooving, phloem necrosis and the complete decay of the tree .
During infection the replication of RNA viruses generate long dsRNA intermediate molecules that triggers the synthesis of small interfering RNAs (siRNAs) . Furthermore, the folded duplex regions of single stranded viral RNAs can also result in siRNA synthesis . These virus-derived siRNAs (vsiRNAs) subsequently regulate viral RNA expression through a process known as RNA silencing. In addition to vsiRNA production, plants’ endogenous small RNA (sRNA) pathways are also affected by viral infection [6–8].
With the introduction of next-generation sequencing the knowledge of sRNA species has been extended beyond the well-characterised miRNA, trans-acting siRNA (tasiRNA) and natural-antisense transcript (NAT) siRNA (nat-siRNA) groups. Although sRNAs were shown to originate from tRNA before, Lee et al.  was the first to illustrate that these molecules were not produced by non-systematic tRNA degradation . Small RNAs associated with tRNAs have been divided into two categories based on the tRNA region they originate from. The first group, called tRNA halves (tsRNA/tiRNA), are derivatives of mature tRNAs cleaved in the anticodon loop, resulting in functional sRNAs of around 28 to 36 nucleotides in size. Enzymes involved in their biogenesis have been identified for humans , yeast  and bacteria , but are still unknown in plants.
Transfer RNA cleaved in the D or T loop give rise to a second group of sRNAs, called tRNA-derived RNA fragments (tRFs). This group can be further divided into sRNAs stemming from (a) the 5′ end of mature tRNAs, (b) the 3′ end of mature tRNAs and (c) the 3′ end of immature tRNAs, called 5′-tRFs, 3′ CCA tRFs and 3′ U tRFs respectively . Several synonyms have been used for the different sub-groups [9, 14].
In this study a next-generation sequencing approach was followed to identify sRNAs that are associated with a latent virus infection in apple plants. In addition to illustrating the vsiRNA profiles associated with an ASGV genetic variant the results from this study demonstrate the involvement of tRNA-derived sRNAs in plant-virus interaction. The lack of differential regulation of miRNAs, phasiRNAs, nat-siRNAs and rasiRNAs in leaf material is also shown.
Results and discussion
sRNA sequencing libraries
vsiRNAs resulting from ASGV infection
Results for the virus-infected NRL sRNA read-mapping against ASGV genomes
GenBank Accession number
Genome size (nt)
Total number of reads mapped
Non-redundant number of reads mapped
Genome coverage (%)
Results for the vsiRNA variant-specific read-mapping
GenBank Accession number
ASGV-infected sample 1
ASGV-infected sample 2
ASGV-infected sample 3
All ASGV-infected samples
Results for the virus-infected BRL sRNA read-mapping against ASGV genomes
GenBank Accession number
Genome size (nt)
Total number of reads mapped
Non-redundant number of reads mapped
Genome coverage (%)
tRNA-derived sRNAs show differential regulation due to ASGV infection
sRNAs, originating from both 5′ and 3′ ends of mature tRNAs, were identified in datasets from both library types (Additional file 1: Table S1 and S2). Additionally, and in agreement with previous studies [23, 26], sRNAs were also identified originating from the central part of tRNAs. These internal species were especially prominent in the cluster of sRNAs (in the BRL data) spawning from tRNA-GlnCTG.
The biogenesis of tRNA-derived sRNAs, as well as the way in which they affect other molecular pathways remains to be elucidated. Earlier reports speculated that tRFs bind, to ribosomes resulting in a down-regulation of gene expression . Through their association with argonaute proteins a possible role in post-transcriptional gene silencing was also suggested . The biological function of the differentially regulated tRNA-derived sRNAs in the current study remains to be determined.
The involvement of other endogenous sRNAs in ASGV infection
Besides the vsiRNAs and tRNA-derived sRNAs involved in ASGV infection, differential expression analysis showed no variation in phasiRNA and miRNA levels as a result of ASGV infection; neither did the nat-siRNAs or rasiRNAs show any change in expression levels (Additional file 2: Table S7 to S17). In addition to their regulatory role during plant development, miRNAs are often linked to stress response. The latent nature of ASGV may therefore explain what seems to be a lack of miRNA involvement during ASGV infection.
In this study next-generation sequencing of sRNAs was used to investigate plant responses to latent virus infection. Two different sRNA libraries were generated per sample. Both datasets illustrated the synthesis of virus-derived sRNAs in response to ASGV infection. Along with earlier reported tRNA-derived sRNAs of more than 30 nt in length, BRL data from this study additionally suggested virus-derived RNAs larger than the well-characterised vsiRNAs of around 21 nt. The vsiRNA profiles varied depending on the method of library preparation used, illustrating the importance of consistency when comparing different samples. Additionally, the results showed that ASGV-infection resulted in a change in the expression of tRNA-derived sRNAs, although the biological function of these sRNAs remains to be elucidated. This study is the first to report on sRNAs involved in ASGV-infection in the domesticated apple.
Sequencing library construction and data preparation
Sample material was collected from three healthy and three asymptomatic ASGV-infected (as confirmed by RT-PCR), greenhouse-grown, M. x domestica cv. ‘Golden Delicious’ (NIVV) seedlings, grafted onto MM.109 rootstocks. The viral status was confirmed by two multiplex RT-PCR reactions described in Menzel et al. . The primers for Apple mosaic virus detection were replaced with those described in Hassan et al. . See Additional file 3: Table S18 for primer information. Total RNA was extracted from mature leaf material using the Plant RNA Reagent Kit (Invitrogen) and used for library (BRL) preparation by means of the TruSeq Small RNA library preparation kit from Illumina. For each sample a second library (NRL) was prepared using the small RNA fraction (17–29 nt) purified from total RNA using a 15% TBE-urea polyacrylamide gel. The final BRL and NRL libraries were size-selected by means of a 3% Pippin Prep cassette (Sage) and a 6% polyacrylamide gel (Invitrogen), respectively, and sequenced on an Illumina HiScan SQ instrument. The software cutadapt (V 1.0)  was used to remove adapter sequences and the reads were filtered for quality (phred score ≥ 20) using FASTX-toolkit (V 0.0.13) . For the NRL, reads less than 17 or longer than 26 nt in length were discarded, while all filtered reads 17 nt and longer were used for the analysis of the BRL data.
Reads from the three NRL virus-infected datasets were combined for vsiRNA analyses. Reads that could map with less than two mismatches onto the apple nuclear, chloroplast or mitochondrial genomes, obtained from the Genome Database for Rosaceae [32, 33], were removed. Bowtie (V 0.12.7)  was used to perform all read-mapping analyses. The filtered reads were then mapped onto six ASGV genomes, allowing only a single mismatch. Similar analyses were performed for the pooled BRL virus-infected samples. Variant-specific reads were identified as those reads that uniquely mapped (using Bowtie) onto one of the six ASGV genomes, when only allowing perfect matches between the sRNA read and the genome.
tRF and tRNA-half identification
Mature tRNA sequences of five angiosperms (Arabidopsis thaliana, Brachypodium distachyon, Medicago truncatula, Oryza sativa and Populus trichocarpa) were retrieved from the PlantRNA database . To identify apple tRFs present, the six NRL datasets were combined and mapped, with Bowtie, onto the retrieved mature tRNA sequences, allowing two mismatches. tRNA-halves were correspondingly identified using the pooled BRL datasets.
Differential expression analysis of apple sRNA species
The standalone differential expression tool of miRanalyzer [36, 37], which implements the R package, DESeq2 , was used to determine variation in sRNA expression levels between the healthy and the ASGV-infected samples. Five distinct sRNA species were investigated using the NRL data, namely miRNAs, phasiRNAs, nat-siRNAs, rasiRNAs and tRFs. The BRL data was used for tRNA-halves differential expression analysis. miRNA analysis was based on miRBase (version 20) [39–42] apple entries, as well as recently predicted novel miRNAs . The phasiRNAs, nat-siRNAs and rasiRNAs analysed were also previously identified , while the tRFs and tRNA-haves were identified during the current study. The phasiRNAs included a group of apple tasiRNAs available on the tasiRNAdb [44–46].
Availability of supporting data
The datasets supporting the results of this article are available in the BioProject repository of the National Centre for Biotechnology Information, BioProject: PRJNA235941 in http://www.ncbi.nlm.nih.gov/bioproject/.
Apple stem grooving virus
Broad range library
- M. x domestica :
Malus x domestica
Narrow rang library
Natural-antisense transcript siRNA
Small interfering RNA
The authors would like to acknowledge Bernard Visser and Michael Hackenberg for bioinformatics support, as well as the National Research Foundation (NRF) for their financial assistance towards this research. Opinions expressed and conclusions arrived at, are those of the authors and are not necessarily to be attributed to the NRF.
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