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

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. 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. 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.


Background
Plant microRNAs (miRNAs) and small interfering RNAs (siRNAs) are two major classes of non-coding small RNAs with 21-24 nucleotides in length that mediate RNA silencing pathways to regulate gene expression and innate immune response [1][2][3][4]. However, miRNA and siRNA are distinct from modes of biogenesis and defense. Specifically, miRNA is primarily generated from a singlestranded precursor that forms a self-complementary stem and loop structure, whereas siRNA is derived from doublestranded RNAs or exogenous viruses. In plants, most miR-NAs are processed specifically by the Dicer-Like 1 (DCL1) protein, and in contarst, siRNAs are cleaved by DCL2, DCL3 and DCL4. After excision, mature miRNAs or siR-NAs are loaded onto the Argonaute (AGO) proteins, with other factors to assemble the RNA-induced silencing complex (RISC), guiding miRNA/siRNA to pair with specific RNA targets to implement the translational repression or silencing [5][6][7][8][9][10][11]. siRNA function is primarily to detect and eliminate invading viruses by targeting the virus-derived single strand RNA (ssRNA). However, miRNAs species usually negatively regulate their target genes via translational inhibition or cleavage of target mRNAs and thus play a pivotal role in a wide variety of biological processes such as plant development, signal transduction, resistance pathways, as well as abiotic and biotic stresses [12][13][14][15][16]. Upon viral infection, miRNAs often exhibit differential expression profiles [17][18][19]. For instance, miRNAs in cotton plants are differentially regulated by infection with Cotton leafroll dwarf virus (CLRDV) and some CLRDV-induced symptoms may be correlated with the deregulation of miRNA and/or epigenetic networks [20]. In N. benthamiana and Arabidopsis, the expression of miR168 and AGO1 mRNA is up-regulated in response to infection by several plant viruses [21][22][23][24]. The same also holds true for soybean mosaic virus (strain G7)-infected soybean plants carrying the resistance gene Rsv1 [19]. As AGO1 protein is a central component of the RISC in the miRNAs/siRNAs-mediated post-transcriptional gene silencing (PTGS) pathways [25], these findings suggest the possible role of miRNA in regulating the innate antiviral silencing pathways in plants.
Pear is an important fruit tree crop cultivated worldwide. China, the world's major producer of pears, has distinctive local pear varieties; however, many pear cultivars are commonly infected with Apple stem grooving virus (ASGV) and Apple chlorotic leaf spot virus (ACLSV), and viral infection dramatically reduces fruit quality [26][27][28][29]. Obtaining virus-free seedlings by heat treatment combined with shoot meristem tip culture is an effective way to control virus diseases in fruit trees [30]. In a previous study, we found that viruses were distributed unevenly in in vitro-grown pear shoots, and that the viral titer apparently decreased in the pear shoot apical meristem in response to high temperature treatment [31]. However, the molecular mechanism underlying the thermotherapy elimination of viruses is not known. To understand the relationship between the high temperature-dependent decrease in viral titer and the miRNA pathways in pear shoots, we used in vitro-grown pear shoots infected with ASGV, a member of the genus Capillovirus [32], as a research material. We sequenced and compared small RNAs prepared from P. pyrifolia shoot meristem tip tissue cultured in vitro at 24°C and at 37°C, a high temperature treatment. The expression levels of viral genomic RNA, miRNAs and mRNAs of their predicted target genes in the shoot meristem tip and base tissues were analyzed to explore the possible roles of miRNA regulation in the high temperaturedependent decrease in virus titer.

Results
Analysis of small RNAs from in vitro-cultured pear shoots infected with ASGV in response to high temperature To identity miRNAs associated with high temperature treatment, small RNA differential expression libraries were constructed from 24°C-and 37°C-treated in vitrogrown pear shoots infected with ASGV and sequenced using high-throughput Solexa sequencing. After removing the low quality reads, 5′ primer contaminants, reads without the 3′ primer, reads with no insert tags, reads containing poly A tags, and reads shorter than 18 nt and longer than 30 nt reads, a total of 22,592,997 and 20,411,254 clean reads were obtained from the meristem tips of in vitro pear shoots cultured at 24°C and 37°C, respectively (Additional file 1). The sequenced clean small RNAs included different categories of exon antisense and sense, intron antisense and sense, rRNA, repeats, tRNA, snRNA, snoRNA, miRNA and other unannotated reads, of which miRNA tags accounted for 9,547,708 ( (Fig. 1a). Interestingly, among the unique sequences, the 24 nt sRNA sequences were most abundant, followed by the 23 nt sRNA sequences, while the 21 nt and 22 nt sequences were present in similar amounts (Fig. 1b). Based on the sizes of miRNAs, the most abundant were 21 nt in length, with a total of 8,963,557 and 10,485,304 reads in the 24°C and 37°C libraries, respectively (Fig. 2a); this relative abundance of 21-nt miRNAs is similar to results reported previously in P. mume [33]. The majority of unique miRNA sequences fell in the range of 21-24 nt in length in both libraries (Fig. 2b), and among them, the 21-nt unique miRNAs were most abundant with 5285 and 5329 reads, followed by the 24-nt miRNAs with 5481 and 4104 reads, while 22-nt and 23-nt sequences were present in similar amounts in the 24°C and 37°C libraries, respectively .
Of the clean reads, the common and specific reads in the 24°C and 37°C libraries from the in vitro-grown pear plants, including the unique reads and total reads, were calculated (Addiitonal file 2). The results showed that 11.65 % total sRNAs (51.78 % unique sRNAs) with an average sequence mean frequency of 1.17 were specifically present in the 24°C library, 6.9 % (32.92 % unique) with an average frequency of 1.09 were found only in the 37°C library, and 81.45 % (15.3 % unique) with an average common sequence frequency of 27.78 were copresent in both libraries (Additional file 2). Taken together the above results suggested that there is a huge difference in the number of reads between the 24°C and 37°C samples.

Characterization of known miRNAs in P. pyrifolia
Known miRNAs in P. pyrifolia were identified by alignment to a designated part of miRBase17.0. One hundred forty-nine known miRNAs in the two P. pyrifolia libraries were obtained and identified after removing miRNAs in which the expression levels were too low to be analyzed for differential expression (Additional file 3). Forty-seven members, belonging to 25 conserved miRNA families, were found in the two P. pyrifolia libraries. The numbers and the read counts for the conserved miRNA families were analyzed (Figs. 3 and 4). A majority of the 25 miRNA families contained 2 members, families miR156 and miR159 each contained 4 members, and the remaining ones including miR162, miR391, miR395, miR397, miR399e, miR403, and miR408 had only one member each. The most abundant miRNA was miR156 with 8,192,170 and 9,689,927 reads in the 24°C and 37°C libraries from P. pyrifolia, respectively, while miR156, miR157, miR397, and miR408 were moderately abundant. These results showed that the miRNA members exhibited significant differential expression levels, in agreenment with findings from other fruit species such as apple and grape [34,35]. In addition to the conserved miRNAs, 102 nonconserved miRNAs were identified, of which miR535 was the most abundant with 438,839 and 413,421 reads in the 24°C and 37°C libraries, respectively (Additional file 3). Identification of novel candidate miRNAs in P. pyrifolia The characteristic hairpin structure of miRNA precursors or detection of the corresponding miRNAs can be used to identify novel miRNAs. Beijing Genomics Institute (BGI) company (Shenzhen city, China) has developed a prediction software, Mireap to predict novel miRNAs by exploring the secondary structure using the mfold web server, the Dicer cleavage sites, and the minimum free energy of the annotated small RNA tags which could be mapped to the pear genome sequence (http:// peargenome.njau.edu.cn) [36][37][38][39][40]. A total of 630 new miRNAs were identified, of which 491 and 530 miRNA candidates were from the 24°C and 37°C libraries, respectively. After removing those miRNAs with expression levels that were too low to be analyzed for differential expression, 141 novel miRNAs were identified, of which 77 were differentially expressed; novel262 had the highest abundance with 833 transcripts per million (TPM), followed by novel345 with 43 TPM in the 24°C library. In the 37°C library, novel19 had the highest abundance at 30 TPM, followed by novel337 and novel540 with 18 and 10 TPM, respectively ( Table 2). The results from this analysis revealed that most of the novel miRNAs were present in relatively low abundance, as indicated by their frequencies, in comparison with the conserved miRNAs. In addition, among the 141 novel miRNAs, 32 had complementary miRNA*s, with precursor lengths ranging from 83 to 349 nt and predicted minimal folding energy (MFE) ranging from −28.6 to −161.4 kal/ mol (Additional file 4, Additional file 5, and Additional file 6). This is in agreement with published criteria for novel miRNA [36][37][38], and indicates that these candidate miR-NAs are most likely to be new miRNA family members in P. pyrifolia. Twenty out of 32 new miRNA/new family members were 21 nt in length, while four, seven, and one miRNA had lengths of 23, 22, and 20 nt, respectively (Additional file 4). Also, 109 miRNAs without miRNA*s detected were identified as candidate miRNAs (Additional file 7); these loci, pre-miRNA sequences and structures, and reads from deep sequencing were also shown in Additional file 6 and Additional file 7. The base bias in the first position among the predicted novel miRNA candidates showed that the majority of these novel miRNA candidates started with a 5′ uridine (U) as shown in Additional file 8. In addition, novel miRNA candidate nucleotide bias at each position were also analyzed (Additional file 9).
Changes in differential abundance levels of miRNAs in response to high temperature in P. pyrifolia To identify high temperature-responsive miRNAs, the normalized expression of miRNAs in the two libraries constructed from the 24°C and 37°C treatments was compared. The miRNAs that showed changes in expression levels >1.0-fold with p-values less than 0.05 in 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 response to high temperature treatment in P. pyrifolia infected with ASGV were presented in Table 2. The results showed that seven known miRNA candidates were differentially expressed, while 77 potentially new miRNA candidates were differentially expressed between the two libraries. Three known miRNAs and 47 novel miRNAs were up-regulated, while four known miRNAs and 30 novel miRNAs were downregulated in response to 37°C treatment. Among the 77 differentially expressed potentially novel miRNAs/ new members, 37 novel miRNA were expressed specifically in response to 37°C, while 22 novel miRNAs  were specifically expressed in the 24°C library ( Table 2).
To validate the existence of the predicted differential expression of miRNAs in the pear shoot meristem tip tissue, four known and eight novel miRNAs that were differentially expressed in the two libraries were analyzed by poly (A) real-time quantitative PCR. The PCR primers were listed in Additional file 10. Gene expression levels were presented as fold-changes in the 37°C treatment shoots relative to expression in the 24°C shoots. The results showed that the expression patterns of four known miRNAs and eight novel miRNAs determined by qRT-PCR were similar to those from the deep sequencing data except for miRNA5519, novel482, and novel566 ( Fig. 5a, b). Results from deep sequencing and real-time PCR demonstrated that 37°C high temperatureresponsive miR397a and miR3627-5p were up-regulated in the shoot meristem tip tissue, while miR477b, novel262, novel177, novel345, novel188, novel197, and novel241 were down-regulated in response to 37°C treatment (Fig. 5a, b).
To investigate the effect of high temperature on viral titer with respect to miRNA pathways in different tissues, the expression levels of miR397a, miR477b, miR3627-5p, miR5519, novel262, novel177, novel345, novel482, novel 566, novel188, novel197, and novel241 were also quantified in the shoot basal tissues treated at 24°C and 37°C using qRT-PCR. The results showed that the 37°C treatment induced 2-16 fold increases in miR397a, miR477b, novel262, novel177, novel482, and novel188 expression levels in shoot basal tissue. Treatment at 37°C also resulted in a 0.32-fold decrease in miR5519 and a 0.5-fold decrease in novel345 expression levels, with no apparent fold-change in novel566 (Fig. 5c). The relative expression levels of four miRNAs (miR477b, novel262, novel177, and novel188) changed significantly in both tissues but in opposite directions. Two miRNAs (miR397a and novel345) changed significantly in the base as compared to the tip (Table 3). Thus, our results show that miRNAs in pear shoots cultured in vitro have different expression profiles in the shoot meristem tip as compared to base tissues, indicating that miRNAs differentially regulated at high temperature exhibit tissue-specific expression patterns.
Prediction of potential targets of differentially expressed miRNAs in P. pyrifolia Hundreds of putative target genes of differentially expressed miRNAs that responded to high temperature treatment in pear were predicted by bioinformatics analysis. Our results showed that P. pyrifolia miRNA targets encoded not only indispensible transcription factors, but also non-transcription factor proteins involved in diverse physiological processes. To investigate the regulatory function of miRNAs in P. pyrifolia infected with ASGV, hundreds of potential target genes for four conserved and 64 novel differentially-expressed miRNAs were predicted by Gene Ontology (GO) analysis. There are three structured ontologies in GO as followings: biological processes, cellular component, and molecular function (Fig. 6). In biological processes, the genes were classified into 19 categories. The top five over-represented GO terms were metabolic process, cellular process, singleorganism process, response to stimulus, and signaling. Nine cellular components were identified, with the top four being cell, cell part, organelle, and membrane. Nine molecular functions were identified, with the most frequent being catalytic activity, binding, transporter activity, and nucleic acid binding transcription factor     activity. These results indicated that the miRNAs have broad functions in gene regulatory networks. In addition, many miRNAs may regulate target genes that are involved in disease resistance and defense. For example, the expression of the putative disease resistance protein targeted by novel166, was up-regulated in response to 37°C treatment by RNA-seq analysis (unpublished). High temperature treatment (37°C) specifically induced expression of novel476 and novel482 miRNAs that target a disease resistance RPP13-like protein and disease resistance protein RGA2-like, respectively. This indicates that the predicted miRNA target genes have important functions in the response to high temperature; however the mechanism behind the miRNAmediated high temperature reduction in viral titer is presently unknown.
The effect of high temperature on ASGV in the shoot meristem tip tissue of in vitro-grown P. pyrifolia To investigate the effects of high temperature on ASGV accumulaiton in in vitro-grown P. pyrifolia shoot meristem tip tissues, the ASGV genomic RNA in the samples "-" indicate no detected data by real-time PCR; "-" indicates that miR3627-5p had no predicted target gene Fig. 6 Gene ontology (GO) analysis of the predicted targets for the differentially expressed miRNAs treated at 24°C (normal growth temperature) and 37°C (high temperature; thermotherapy) was quantified by real-time PCR to detect the cp gene. It was found that in comparison with the control, ASGV was reduced to 0.6fold and to 0.25-fold after 37°C treatment for 1 and 5days, respectively (Fig. 7). Apparantly, the ASGV titer in the meristem tip tissue decreased along with increasing treatment times at high temperature.
Spatial expression patterns of potential miRNA-mediated target genes in response to high temperature in P. pyrifolia shoots To examine if high temperature-responsive miRNAs can regulate the expression of their target genes, the expression levels of selected miRNAs and their target mRNAs were analyzed from tip and base tissues of pear shoots.
The changes in spatial expression patterns of laccase-4like, DELLA protein RGL1-like, proteasome subunit alpha type-4, an ABC transporter A family member, NAC domain-containing protein 72-like, 2-oxoglutarate dehydrogenate, mitochondrial-like, disease resistance protein RGA2-like, E3 ubiquitin-protein ligase UPL1like, and squamosa promoter-binding-like Protein13A, as predicted targets of miR397a, miR477b, miR5519, novel262, novel177, novel345, novel482, novel566, and novel188, respectively, were quantified using qRT-PCR (Fig. 8). The correlations between the expression levels of these miRNAs and their target mRNAs in pear shoot tip and base tissues in response to high temperature were shown in Table 3.
In shoot apices, negative correlations were observed for miR397a, miR477b, miR5519, novel262, novel177, novel345, novel482, and novel188 and their corresponding putative target genes, except for novel566. Putative target gene DELLA protein RGL1-like (targeted by mi R477b), proteasome subunit alpha type-4 (by miR5519), ABC transporter A family member (by novel262), NAC domain-containing protein 72-like (by novel177), 2-oxoglutarate dehydrogenate, mitochondrial-like (by nove l345), disease resistance protein RGA2-like (by nove l482), and squamosa promoter-binding-like Protein13A (by novel188) were all up-regulated with relative quantitive (RQ) values of 4.43,1.32, 1.23, 3.66, 1.8, 1.42, and 2.4 in the 37°C treatment, respectively. We also analyzed expression patterns of several miRNA-mediated target genes in shoot base tissue. The results showed that the expression levels of target gene Laccase-4-like increased at 37°C, with RQ 1.22. Expression of ABC transporter A family member and NAC domain-containing protein 72like also increased at 37°C, with RQ values of 1.47 and 2.64, respectively. mRNA levels of 2-oxoglutarate dehydrogenase and squamosa promoter-binding-like Pro-tein13A at 37°C also increased 1.04 and 2.6-fold, respectively. This shows that the changes in mRNA levels in stem base tissue and in shoot tip tissue are different. In addition, the other target genes showed the opposite expression patterns in base tissue compared with tip tissues (Fig. 8 and Table 3).

Discussion
High-throughput sequencing of P. pyrifolia small RNAs In this study, millions of small RNA reads from libraries prepared from ASGV-infected P. pyrifolia shoots grown at 24°C and 37°C were obtained by high-throughput sequencing. The pear genome sequence, released in 2012, was used for annotating the specific miRNAs of P. pyrifolia [40]. The most abundant small RNA reads in the two libraries prepared from the P. pyrifolia samples were 21 nt in length, followed by 24 nt, while 24 nt reads were predominant in the unique small RNA reads (Figs. 1 and 2). This is different from reports in other plant species in which 24 nt sRNAs are more abundant, such as in the wheat response to powdery mildew infection and heat stress [41,42]. The ratio of sRNA total reads/unique sequences in the 24-nt class was 2.3 and 2 in the 24°C and 37°C libraries, respectively, while the ratios in the 21-nt We observed that the 21-nt small RNAs increased and the 24-nt small RNAs decreased in abundance in response to 37°C treatment, and the 21 nt miRNAs were more abundant in the 37°C library than in the 24°C library. These observations demonstrate that the expression of miRNAs and siRNAs is significantly altered by the high temperature treatment, and such dynamic small RNA profiles may have important functions in suppressing viral infection under high temperature treatment.
Possible functional roles of miRNAs in suppressing viral infection in high temperature-treated P. pyrifolia In the P. pyrifolia shoot meristem tip, seven known and 77 potentially novel miRNAs were shown to be differentially expressed in response to high temperature (37°C) treatment and the predicted target genes of these differentially expressed miRNAs were also identified ( Table 2 and Fig. 6). These datas provide valuable insight information about the complex miRNA-mediated regulatory networks in pear shoot meristem tips infected with ASGV in response to high temperature treatment. To the best of our knowledge, this is the first report of high temperature-altered miRNAs that are involved in viral infection.
miRNAs may play important roles in host resistance against abiotic stress and biotic pathogens. For example, down-regulation of miR398 in A. thaliana up-regulates its target gene CSD encoding Cu/Zn superoxide dismutase, a known enzyme that is related to defense response against Pseudomonas syringae [3,16,43,44]. In addition, it has also been reported that infections by Tobacco mosaic virus and Potato virus Y lead to changes in miRNA expression levels that affect the ability of plants to resist disease. These previous studies have demonstrated that miRNAs can regulate key genes in disease resistance pathways to affect viral infections [13,17,18,45,46]. In this study, we identified a number of predicted miRNA-target genes from P. pyrifolia that are possibly involved in disease resistance and defense in the high temperature-induced reduction in ASGV titers  Table 3). For instance, Laccase targeted by miR397a is involved in the formation of the plant cell wall, and plays an important role in the defense response [47]. DELLA protein RGL1-like, TIR-NBS-LRR, and RGA2-like predicted to be targeted by miR-NA477b, novel421 and novel482, respectively are also known as disease resistance proteins. Interestingly, we found that the targets of novel177 severely downregulated in high temperature include NAC transcription factor, heat shock factor protein HSF8-like, and E3 ubiquitin-protein ligase COP1-like, which suggests that novel177 is involved in a complicated miRNA regulatory network. The computational analysis combined with the qRT-PCR results showed that NAC is negatively regulated by novel177 (Figs. 5 and 8). Numerous members of the NAC gene family have been shown to respond to biotic and abiotic stresses which may activate the plant defense response [48][49][50][51][52]. In this study, we found that significantly repressed expression of novel177 significantly up-regulated NAC expression in the shoot meristem tip of P. pyrifolia with low ASGV titer at 37°C as compared to 24°C ( Fig. 8 and Table 3). In Arabidopsis, NAC1, regulated by miRNA164, is crucial for the formation of boundaries between meristems and emerging organ primordia, and affects auxin metabolism in the auxin signaling pathway [53][54][55]. It is possible that up-regulated NAC accelerates the growth and developmental process of the shoot meristem tip tissue, and inhibits the moevment of ASGV into the newly divided tip cells in an as-yet unknown manner during high temperature, leading to the reduced viral titer in the shoot meristem tip [56]. Based on above disucssion, it is very likely that the mixed action of these miRNAmediated target genes contributes to the thermoelimination of ASGV in pear. Indeed, high temperature can activate many highly conserved biological and adaptive responses that are regulated by miRNAs, which exhibit protective functions for the induction of the disease resistance response in plants [41,[56][57][58]. Future work is aimed at elucidation of the mechanistic details of these miRNA-regulated defense genes in inibiting viral infection at high temperature.

Conclusions
This is the first report of miRNAs differentially expressed at 24°C and 37°C in the meristem tip of in vitro-grown pear shoots infected with ASGV. Using Illumina highthroughput sequencing, the numbers and types of miR-NAs were systematically identified from in vitro-grown P. pyrifolia shoot meristem tip tissue at 24°C and 37°C. This was done in combination with qRT-PCR to explore the interaction between miRNA-regulated pear genes and ASGV in response to high temperature treatment. A total of seven known and 77 novel miRNAs were found to be expressed differentially in response to high temperature. Putative target genes were predicted and then annotated by GO databases to explore predicted gene functions. Comparative analysis of expression levels in relation to virus titer, miRNA and miRNA-mediated target gene expression patterns were performed to reveal the role of miRNA regulation on the decrease in virus titer from the shoot meristem tip observed under conditions of high temperature. Our research provided valuable information for further exploration of the function of these miRNAs/ mRNA in associaiton with the reduced viral titer in meristem tip of P. pyrifolia shoots inducible to high temperature treatment.

Plant materials
P. pyrifolia cv. 'Jinshui no. 2' , a widely grown cultivar in central and southern China, was used in the experiments. The establishment of in vitro P. pyrifolia cv. 'Jinshui no. 2' shoots in culture was performed as reported [59]. The presence of ASGV in each of the different explants was determined by reverse transcrption (RT)-PCR. In vitro shoots about 1 cm in length were transferred to freshly prepared MS medium and incubated in a thermotherapy chamber at 37°C with a day/ night regime of 16 h light at an intensity of 1500 lux and 8 h dark. Non-treated shoots infected with ASGV were kept at 24°C as controls. Six shoots of 5 mm in length were collected from meristem tip and treated at 37°C for 1 and 5 days, with three replications per treatment. Untreated meristem tip shoots (5 mm) maintained at 24°C for 1 and 5 days were used as controls. All samples were frozen in liquid nitrogen immediately and stored at −80°C prior to extraction of total RNA.

Small RNA library construction and Illumina sequencing
To construct the small RNA libraries, total RNA was extracted from the pear tissue samples treated for 1 and 5 days at 24°C and 37°C using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Mixed samples containing equal amounts of total RNA from the 1-and 5-day treatments at 37°C, were designated as T37, and a mixed sample containing equal amounts of the two treatments at 24°C was the T24 control. The integrity and concentration of the total RNA samples were checked using a NanoDrop spectrophotometer and Agilent 2100 Bioanalyzer. High quality samples were used to construct small RNA library for sequencing on the Illumina Hiseq 2000 instrument at Beijing Genomics Institute (BGI) company (Shenzhen, China).

Deep sequencing data analyses
All raw sequencing data was processed using the SOAP software (BGI Company) to obtain clean reads from each library as follows: except for low quality reads, reads with 5′ primer contaminants, reads without a 3′ primer, reads with no insert tags, reads with poly A tags, reads shorter than 18 nt and longer than 30 nt. Firstly, the sequences homologous to non-coding RNAs, including rRNAs, tRNAs, snRNAs, snoRNAs and siRNA were removed from the matched sequences through BLASTn searches using the NCBI Genebank (http:// www.ncbi.nlm.nih.gov/blast/Blast.cgi/) and Rfam databases (http://www.sanger.ac.uk/Software/Rfam/). The remaining sequences were aligned to the pear genome sequence (http://peargenome.njau.edu.cn) with known miRNAs from miRBase17 and those matched to the pear genome shotgun-sequence assemblies were kept for known miRNA identification. Mireap software developed by BGI to predict novel miRNAs based on certain rules for stem-loop hairpins described [36][37][38][39], can be accessed from the following link: http://sourceforge.net/projects/ mireap/.

Differential expression analysis of miRNAs in response to high temperature
The expression levels of miRNAs in the 24°C (control) and 37°C (high temperature) treatments from in vitrogrown pear shoots were visualized by plotting the Log2ratio of T37/T24. The procedures used were as follows: (1) The expression of miRNAs in the 24°C and 37°C libraries to get the number of transcripts per million (TPM) were normalized. Normalized expression = actual miRNA count/Total number of clean reads*1000000. In addition, the normalized expression of the miRNA was corrected to 0.01, when the miRNA gene count was zero.
(2) The fold-change and P-value from the normalized expression were calculated. Fold-change = log 2 (T37 treatment/T24 control); the P-value was calculated as reported previously [60,61]. The expression levels of miRNAs with fold changes >1.0-fold and P -values < 0.05 were considered to be responsive to high temperature treatment in P. pyrifolia shoots.
Prediction of potential target genes for miRNA candidates in P. pyrifolia Differentially expressed miRNAs were used as query sequences in BLASTn searches against the pear unigene database (http://peargenome.njau.edu.cn). The criteria for predicted mRNA target genes by alignment with each miRNA were based on those previously reported [62,63].

Real-time PCR analysis of miRNA and target gene expression
Real-time PCR was used to confirm the miRNA expression levels obtained from the high-throughput sRNA sequencing. Total RNAs were extracted from the samples treated for 1 and 5 days at 24°C and 37°C, respectively, using CTAB methods as described [64]. For miRNA, poly (A) tails were first added to the 3′ ends, and cDNA was then synthesized by RT using the One Step Prime-Script miRNA cDNA Synthesis Kit with a special oligo-dT adaptor according to the manufacturer's instructions (TaKaRa, Dalian, China). The cDNA products were used as templates for quantitative PCR analysis, which was performed on a CFX96 Real-time System (BIO-RAD, USA) using SYBR Premix Ex Taq II (TaKaRa). All specific miRNA forward primers were designed based on the mature miRNA sequence, while the reverse primers were the adaptor sequence provided in One Step Prime-Script miRNA cDNA Synthesis Kit (Additional file10). In addition, qRT-PCR was used to analyze the expression of target genes cleaved by miR-NAs and ASGV cp mRNAs, and the primers are also listed in Additional file 10. Total RNA digested by DNAase as template was reverse-transcribed using M-MLV reverse transcriptase (Promega, USA) and 6-base random primers to obtain cDNA. Each RT reaction contained 2.5 μl diluted cDNA, 12.5 μl of the SYBR Premix Ex Taq II (2×) PCR mixture, 1 μl of each 5 mM primer, and sterile water to a final volume 25 μl. The Actin gene was used as the internal reference gene. The specificity of the primers was verified by analysis of the melting curves from a thermal denaturing cycle of 60-95°C with 1°C increments applied for 1 s. All reactions were run in triplicate. The real-time PCR program conditions were as the follows: 95°C for 30s, followed by 40 cycles of 95°C for 5 s, and 60°C for 30 s. The values for each miRNA/mRNA in the T24 sample were set as 1, and miRNA/mRNA relative expression level changes were calculated by a comparative C T method (ΔΔC T ) using the formula 2 -ΔΔCt [65].