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. 2015 Nov 16:16:945.
doi: 10.1186/s12864-015-2126-8.

Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing

Juan Liu  1   2   3   4 , XueJiao Zhang  2   5 , FangPeng Zhang  1   2   3   4 , Ni Hong  1   2   3   4 , GuoPing Wang  1   2   3   4 , Aiming Wang  6 , LiPing Wang  7   8   9   10
Affiliations

Identification and characterization of microRNAs from in vitro-grown pear shoots infected with Apple stem grooving virus in response to high temperature using small RNA sequencing

Juan Liu et al. BMC Genomics. .

Abstract

Background: MicroRNAs (miRNAs) have functions in diverse biological processes such as growth, signal transduction, disease resistance, and stress responses in plants. Thermotherapy is an effective approach for elimination of viruses from fruit trees. However, the role of miRNAs in this process remains elusive. Previously, we showed that high temperature treatment reduces the titers of Apple stem grooving virus (ASGV) from the tips of in vitro-grown Pyrus pyrifolia plants. In this study, we identified high temperature-altered pear miRNAs using the next generation sequencing technology, and futher molecularly characterized miRNA-mediated regulaton of target gene expression in the meristem tip and base tissues of in vitro-grown, ASGV-infected pear shoots under different temperatures.

Results: Using in vitro-grown P. pyrifolia shoot meristem tips infected with ASGV, a total of 22,592,997 and 20,411,254 clean reads were obtained from Illumina high-throughput sequencing of small RNA libraries at 24 °C and 37 °C, respectively. We identified 149 conserved and 141 novel miRNAs. Seven conserved miRNAs and 77 novel miRNAs were differentially expressed at different temperatures. Target genes for differentially expressed known and novel miRNAs were predicted and functionally annotated. Gene Ontology (GO) analysis showed that high-ranking miRNA target genes were involved in metabolic processes, responses to stress, and signaling, indicating that these high temperature-responsive miRNAs have functions in diverse gene regulatory networks. Spatial expression patterns of the miRNAs and their target genes were found to be expressed in shoot tip and base tissues by qRT-PCR. In addition, high temperature reduced viral titers in the shoot meristem tip, while negatively regulated miRNA-mediated target genes related to resistance disease defense and hormone signal transduction pathway were up-regulated in the P. pyrifolia shoot tip in response to high temperature. These results suggested that miRNAs may have important functions in the high temperature-dependent decrease of ASGV titer in in vitro-grown pear shoots.

Conclusions: This is the first report of miRNAs differentially expressed at 24 °C and 37 °C in the meristem tip of pear shoots infected with ASGV. The results of this study provide valuable information for further exploration of the function of high temperature-altered miRNAs in suppressing viral infections in pear and other fruit trees.

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Figures

Fig. 1
Fig. 1
Length distribution of small RNA reads in the 24 °C and 37 °C libraries constructed from in vitro-grown shoots of P. pyrifolia. Length distribution of sRNA reads (a) and unique sequences (b) in the two libraries
Fig. 2
Fig. 2
Characterization of miRNAs in the 24 °C and 37 °C in vitro pear shoot libraries. Length distribution of miRNA reads (a) and unique sequences (b) in the two libraries
Fig. 3
Fig. 3
Number of miRNA members identified in each conserved miRNA family in the small RNA libraries
Fig. 4
Fig. 4
Count numbers of identified miRNAs in each conserved miRNA family in the 24 °C and 37 °C in vitro pear shoot libraries
Fig. 5
Fig. 5
Relative miRNA expression profiles determined in in vitro pear shoot tips in the 24 °C and 37 °C libraries by deep sequencing (a) and quantitative real-time PCR (b), and in base tissue by qRT-PCR (c)
Fig. 6
Fig. 6
Gene ontology (GO) analysis of the predicted targets for the differentially expressed miRNAs
Fig. 7
Fig. 7
ASGV virus titer in tip meristem tissues of in vitro pear shoots determined by qRT-PCR analysis of the ASGV cp gene in the 24 °C and 37 °C small RNA libraries
Fig. 8
Fig. 8
miRNA target gene expression profiles in the shoot tip (a) and base tissues (b) of in vitro-grown pear shoots determined by qRT-PCR
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