title: 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.
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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Wuhan, Hubei, 430070, P. R. China.|College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P. R. China.|National Indoor Conservation Center of Virus-Free Germplasms of Fruit Crops, Wuhan, Hubei, 430070, P. R. China.|Lab of Key Lab of Plant Pathology of Hubei Province, Wuhan, Hubei, 430070, P. R. China.
authors.forename: Juan
authors.initials: J
authors.lastname: Liu
authors.affiliation: College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P. R. China.|Shihezi University, Shihezi City, Xinjiang Uyghur Autonomous Region, 832003, P. R. China.
authors.forename: XueJiao
authors.initials: X
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Wuhan, Hubei, 430070, P. R. China.|College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P. R. China.|National Indoor Conservation Center of Virus-Free Germplasms of Fruit Crops, Wuhan, Hubei, 430070, P. R. China.|Lab of Key Lab of Plant Pathology of Hubei Province, Wuhan, Hubei, 430070, P. R. China.
authors.forename: FangPeng
authors.initials: F
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Wuhan, Hubei, 430070, P. R. China.|College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P. R. China.|National Indoor Conservation Center of Virus-Free Germplasms of Fruit Crops, Wuhan, Hubei, 430070, P. R. China.|Lab of Key Lab of Plant Pathology of Hubei Province, Wuhan, Hubei, 430070, P. R. China.
authors.forename: Ni
authors.initials: N
authors.lastname: Hong
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Wuhan, Hubei, 430070, P. R. China.|College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P. R. China.|National Indoor Conservation Center of Virus-Free Germplasms of Fruit Crops, Wuhan, Hubei, 430070, P. R. China.|Lab of Key Lab of Plant Pathology of Hubei Province, Wuhan, Hubei, 430070, P. R. China.
authors.forename: GuoPing
authors.initials: G
authors.lastname: Wang
authors.affiliation: Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada, London, N5V 4T3, ON, Canada.
authors.forename: Aiming
authors.initials: A
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Wuhan, Hubei, 430070, P. R. China. wlp09@mail.hzau.edu.cn.|College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei, 430070, P. R. China. wlp09@mail.hzau.edu.cn.|National Indoor Conservation Center of Virus-Free Germplasms of Fruit Crops, Wuhan, Hubei, 430070, P. R. China. wlp09@mail.hzau.edu.cn.|Lab of Key Lab of Plant Pathology of Hubei Province, Wuhan, Hubei, 430070, P. R. China. wlp09@mail.hzau.edu.cn.
authors.forename: LiPing
authors.initials: L
authors.lastname: Wang
mesh_terms: D052677:Flexiviridae / Q000502:physiology*; D020869:Gene Expression Profiling; D017343:Genes, Plant; D059014:High-Throughput Nucleotide Sequencing; D006358:Hot Temperature; D066298:In Vitro Techniques; D035683:MicroRNAs / Q000235:genetics*; D010935:Plant Diseases / Q000235:genetics* / Q000821:virology*; D018547:Plant Stems; D031989:Pyrus / Q000235:genetics* / Q000821:virology*; D018749:RNA, Plant / Q000235:genetics*; D017423:Sequence Analysis, RNA
chemical_list: D035683:MicroRNAs; D018749:RNA, Plant