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. 2016 Oct 6;13(1):166.
doi: 10.1186/s12985-016-0625-0.

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

Juan Liu  1   2 , XueJiao Zhang  3 , YueKun Yang  1   2 , Ni Hong  1   2 , GuoPing Wang  1   2 , Aiming Wang  4 , LiPing Wang  5   6
Affiliations

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

Juan Liu et al. Virol J. .

Abstract

Background: Heat 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.

Results: At 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.

Conclusions: Collectively, 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.

Keywords: Apple stem grooving virus; Argonaute (AGO); Dicer-like (DCL); Gene silencing; High temperature; Pyrus pyrifolia; RNA dependent RNA polymerase (RDRs); RT-qPCR; Virus-derived small interfering RNA (vsiRNA).

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Figures

Fig. 1
Fig. 1
ASGV-Js2 genome analysis. a Diagram showing the organization of the ASGV-Js2 genome; b Phylogenetic analysis of ASGV full-length genomic sequences from the ASGV-Js2 isolate used in this study with 17 previously-reported isolates. Isolate names are followed by the GenBank accession number, the plant host, and the country of origin in parentheses. The tree was reconstructed by the Neighbour-joining method (NJ) with 1,000 bootstrap replicates, and bootstrap values >50 % are shown at branch nodes. Bars represent 0.02 substitutions per site
Fig. 2
Fig. 2
Analysis of the ASGV-derived vsiRNA population in P. pyrifolia shoots. a Distribution of ASGV-Js2-derived vsiRNA lengths from P. pyrifolia shoots cultured in vitro at 24 °C and 37 °C. The numbers of siRNAs (18–26 nt) in the 24 and 37 °C libraries are shown in gray and blank, respectively. b Distribution of the numbers of sense and antisense vsiRNAs reads in the 24 and 37 °C libraries. The positive and negative vsiRNAs reads mapped to strands of the virus are represented in gray and blank, respectively. Histograms represent the numbers of sense and antisense vsiRNA reads in each size class
Fig. 3
Fig. 3
Relative frequencies of the 5'-terminal nucleotide in pear vsiRNAs. The positive- and negative-strand vsiRNAs were derived from ASGV-Js2-infected in vitro-grown P. pyrifolia shoots at 24 °C (a) and 37 °C (b)
Fig. 4
Fig. 4
Characterization of the vsiRNA 5'-terminal nucleotides. The relative abundance of the four 5'-terminal nucleotides are shown for the 20 to 24 nt vsiRNAs. The genomic and anti-genomic ASGV-derived vsiRNAs were from in vitro-grown P. pyrifolia shoot tips at 24 °C (a) and 37 °C (b)
Fig. 5
Fig. 5
Profiles of variant-specific sRNA reads along the ASGV-Js2 genome. The genomic positions of vsiRNA reads mapped to the ASGV-Js2 genome are shown. a and b show the profiles of the ASGV-derived vsiRNAs mapped to the ASGV-Js2 genome from the 24 and 37 °C libraries, respectively. Reads that mapped to the positive or negative strands of the ASGV-Js2 genome are represented in green and red, respectively
Fig. 6
Fig. 6
Profiles of vsiRNAs derived from the positive and negative strands of the ASGV genome by qRT-PCR. Error bars indicate standard deviations. Asterisks represent significant differences
Fig. 7
Fig. 7
Relative accumulation of ASGV-Js2 genomic RNA and MP subgenomic RNA in tip tissues of in vitro-grown pear shoots in response to 37 °C treatment. Error bars indicate standard deviations. Asterisks represent significant differences
Fig. 8
Fig. 8
Phylogenetic relationships between the PpDCL2,4, PpAGO1,2,4, and PpRDR1 proteins of P. pyrifolia with their homologues in Pyrus bretschneideri, Malus, Oryza sativa, Nicotiana, and Arabidopsis species. a, b, and c are unrooted Neighbor-joining trees constructed with predicted protein sequences for Dicer-like, Argonaute, and RNA-dependent RNA polymerases from this study and homologous sequences from other isolates deposited into GenBank (gene name followed by GenBank accession number). Statistical analysis was performed with 1,000 bootstrap replicates, and bootstrap values >50 % are shown at branch nodes. Bars represent 0.1 substitutions per site. The predicted P. pyrifolia DCL, AGO, and RDR proteins are shown in red in each group. The abbreviations for the AGO, DCL, and RDR proteins used in the phylogenetic trees are as the follows: Pb, Pyrus bretschneideri; Pp, Pyrus pyrifolia; Md, Malus domestic; Os, Oryza sativa; Nb, Nicotiana benthamiana; Nt, Nicotiana thaliana; At, Arabidopsis thaliana
Fig. 9
Fig. 9
Relative expression levels of PpDCL2 and PpDCL24 (a), PpAGO1, PpAGO2, and PpAGO4 (b) and PpRDR1 (c) genes were determined from total RNA isolated from ASGV-infected and uninfected P. pyrifolia shoot tips at 24 and 37 °C by RT-qPCR. Error bars indicate standard deviations. Bars in each histogram labeled with the same letters are not significantly different
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References

    1. Hamilton AJ, Baulcombe DC. A species of small antisense RNA in posttranscriptional gene silencing in plants. Science. 1999;286:950–952. doi: 10.1126/science.286.5441.950. - DOI - PubMed
    1. Ding SW, Voinnet O. Antiviral immunity directed by small RNAs. Cell. 2007;130:413–426. doi: 10.1016/j.cell.2007.07.039. - DOI - PMC - PubMed
    1. Miozzi L, Gambino G, Burgyan J, Pantaleo V. Genome-wide identification of viral and host transcripts targeted by viral siRNAs in Vitis vinifera. Mol Plant Pathol. 2013;14:30–43. doi: 10.1111/j.1364-3703.2012.00828.x. - DOI - PMC - PubMed
    1. Schuck J, Gursinsky T, Pantaleo V, Burgyán J, Behrens SE. AGO/RISC mediated antiviral RNA silencing in a plant in vitro system. Nucleic Acids Res. 2013;41:5090–5103. doi: 10.1093/nar/gkt193. - DOI - PMC - PubMed
    1. Baumberger N, Baulcombe DC. Arabidopsis ARGONAUTE1 is an RNA slicer that selectively recruits microRNAs and short interfering RNAs. Proc Natl Acad Sci U S A. 2005;102:11928–11933. doi: 10.1073/pnas.0505461102. - DOI - PMC - PubMed

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