Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses

The complete nucleotide sequence of an Indian isolate of Apple chlorotic leaf spot virus (ACLSV) was determined and found to be 7,525 nt in length. The genome organization was similar to known isolates of ACLSV, encoding three ORFs. Comparisons indicated high sequence variability among known isolates with overall nucleotide sequence identities of 80 to 84%. A striking variable region was identified among the replicase protein upstream of the RNA‐dependent RNA polymerase (aa 1510–1590), which showed a 41–43% match with the corresponding region in other isolates. Phylogenetic analysis at the nucleotide level clustered the isolates into three groups, without any relation to geographical origin. Recombination analysis showed that the isolate is a recombinant with recombination sites spread throughout the genome, especially in the polymerase gene region (nt 4700–5400). Most recombination sites were bordered by an upstream region (5′) of GC‐rich and downstream region (3′) of AU‐rich sequences of similar length. Correlation of recombination site with host type is discussed, and it was found that there were more interlineage recombinations in the apple host compared with intralineage recombinations.

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“…This conserved motif is the transcriptional start site for both subgenomic RNAs, as reported by Komatsu et al. ().…”

Section: Resultssupporting

confidence: 67%

Molecular Characterization and Recombination Analysis of the Complete Genome of Apple Chlorotic Leaf Spot Virus

Dhir

Zaidi

Hallan

2013

Journal of Phytopathology

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“…The increase in vsiRNAs production toward the 3′ end of the genome has previously been ascribed to the presence of viral subgenomic RNAs (sgRNAs) [17, 18]. Both the ASGV movement and coat proteins are expressed from 3′ sgRNAs [19–21] and may explain the increase in vsiRNA originating from the 3′ end. The non-redundant reads were also plotted onto the ASGV genomes (Figure 5).…”

Section: Resultsmentioning

confidence: 99%

High-throughput sequencing reveals small RNAs involved in ASGV infection

et al. 2014

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BackgroundPlant 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.ResultssRNA 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.ConclusionsThis 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.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-568) contains supplementary material, which is available to authorized users.

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“…ORF2 is embedded within the ORF1 and encodes a movement protein (MP) of approximately 36 kDa [ 27 ]. MP and CP may be produced through the 3′-coterminal subgenomic RNAs (sgRNAs), and CP expression from sgRNA is essential for ASGV systemic infection in the host [ 28 , 29 ]. Phylogenetic analysis of 16 ASGV full-length genomic sequences clusters them into two groups with no correlations to host and geographical origins [ 30 , 31 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of virus-derived small interfering RNAs in Apple stem grooving virus-infected in vitro-cultured Pyrus pyrifolia shoot tips in response to high temperature treatment

Liu

Zhang

Yang

et al. 2016

Virol J

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BackgroundHeat treatment (known as thermotherapy) together with in vitro culture of shoot meristem tips is a commonly used technology to obtain virus-free germplasm for the effective control of virus diseases in fruit trees. RNA silencing as an antiviral defense mechanism has been implicated in this process. To understand if high temperature-mediated acceleration of the host antiviral gene silencing system in the meristem tip facilitates virus-derived small interfering RNAs (vsiRNA) accumulation to reduce the viral RNA titer in the fruit tree meristem tip cells, we used the Apple stem grooving virus (ASGV)–Pyrus pyrifolia pathosystem to explore the possible roles of vsiRNA in thermotherapy.ResultsAt first we determined the full-length genome sequence of the ASGV-Js2 isolate and then profiled vsiRNAs in the meristem tip of in vitro-grown pear (cv. ‘Jinshui no. 2’) shoots infected by ASGV-Js2 and cultured at 24 and 37 °C. A total of 7,495 and 7,949 small RNA reads were obtained from the tips of pear shoots cultured at 24 and 37 °C, respectively. Mapping of the vsiRNAs to the ASGV-Js2 genome revealed that they were unevenly distributed along the ASGV-Js2 genome, and that 21- and 22-nt vsiRNAs preferentially accumulated at both temperatures. The 5′-terminal nucleotides of ASGV-specific siRNAs in the tips cultured under different temperatures had a similar distribution pattern, and the nucleotide U was the most frequent. RT-qPCR analyses suggested that viral genome accumulation was drastically compromised at 37 °C compared to 24 °C, which was accompanied with the elevated levels of vsiRNAs at 37 °C. As plant Dicer-like proteins (DCLs), Argonaute proteins (AGOs), and RNA-dependent RNA polymerases (RDRs) are implicated in vsiRNA biogenesis, we also cloned the partial sequences of PpDCL2,4, PpAGO1,2,4 and PpRDR1 genes, and found their expression levels were up-regulated in the ASGV-infected pear shoots at 37 °C.ConclusionsCollectively, these results showed that upon high temperature treatment, the ASGV-infected meristem shoot tips up-regulated the expression of key genes in the RNA silencing pathway, induced the biogenesis of vsiRNAs and inhibited viral RNA accumulation. This study represents the first report on the characterization of the vsiRNA population in pear plants infected by ASGV-Js2, in response to high temperature treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0625-0) contains supplementary material, which is available to authorized users.

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Infection of capilloviruses requires subgenomic RNAs whose transcription is controlled by promoter-like sequences conserved among flexiviruses

Cited by 24 publications

References 19 publications

Molecular Characterization and Recombination Analysis of the Complete Genome of Apple Chlorotic Leaf Spot Virus

Molecular Characterization and Recombination Analysis of the Complete Genome of Apple Chlorotic Leaf Spot Virus

High-throughput sequencing reveals small RNAs involved in ASGV infection

Characterization of virus-derived small interfering RNAs in Apple stem grooving virus-infected in vitro-cultured Pyrus pyrifolia shoot tips in response to high temperature treatment

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