Genome-wide expression profiling of microRNAs in poplar upon infection with the foliar rust fungus Melampsora larici-populina

Chen and Cao BMC Genomics (2015) 16:696 DOI 10.1186/s12864-015-1891-8 RESEARCH ARTICLE Open Access Genome-wide expression profiling of microRNAs in poplar upon infection with the foliar rust fungus Melampsora larici-populina Min Chen and Zhimin Cao* Abstract Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate the gene expression of target mRNAs involved in plant growth, development, and abiotic stress and pathogen responses. Previous studies have reported miRNAs in Populus that respond to abiotic stresses, such as cold, heat, drought, flooding, high salt and mechanical stress. However, little is known about the regulatory roles of these molecules in the Populus response to the stress of foliar rust fungal infection. Here, we identified the miRNA profiles of Populus after inoculation with Melampsora larici-populina using high-throughput sequencing and bioinformatics analysis. Quantitative real-time PCR (qRT-PCR) was used to validate the expression levels of 10 miRNAs. Results: A total of 90 known miRNAs belonging to 42 families and 378 novel miRNAs were identified from three small-RNA libraries of Populus szechuanica infected with M. larici-populina isolates Sb052 and Th053 and a control. Comparative analysis revealed that the expression of 38 known miRNAs and 92 novel miRNAs in P. szechuanica after infection with different rust fungus isolates showed significant differences, and more miRNAs were suppressed during rust infection. Among the differentially expressed miRNAs, 7 known and 20 novel miRNAs were relevant to the rust fungus infection, and according to KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathway analysis, these miRNAs primarily regulate genes encoding disease-resistance proteins, serine/threonine protein kinases, transcription factors, and related proteins. QRT-PCR analysis indicated that most miRNAs were up-regulated in the Sb052 library and down-regulated in the Th053 library at 48 h post-inoculation (hpi). Conclusions: These results demonstrate that the expression of miRNAs was altered in poplar under stress associated with M. larici-populina infection, and different temporal dynamics were observed in incompatible and compatible libraries. These findings suggest important roles for miRNA regulation in Populus upon infection with foliar rust fungus. Keywords: Populus szechuanica, MicroRNA, High-throughput sequencing, Melampsora larici-populina Background MiRNAs, small endogenous non-coding RNAs approximately 21–24 nucleotides (nt) in length, play an important role in regulating gene expression at the post-transcriptional level [1]. A large number of miRNAs have recently been identified in plants, and numerous miRNAs have been entered into the miRBase. miRNAs are involved in regulating growth, development, hormone balance, floral morphogenesis, reproductive * Correspondence: College of Forestry, Northwest A & F University, Yangling, Shaanxi 712100, People’s Republic of China performance, and biotic and abiotic stress responses. Upon nutritional deficiency, miRNAs regulate the metabolic balance of phosphorus, sulphur and copper in plants [2–5]. Under environmental stresses, including drought, flooding, salt, cold and heat, stress-regulated miRNAs in plants confer resistance to the extreme conditions [6]. In addition, the expression of miRNAs can alter in plants in response to biotic stresses, such as fungi, bacteria, viruses, or insects [7–16]. The genome of P. trichocarpa is small, and these plants reach reproductive maturity in a relatively short time [17, 18]. Thus, Populus is a useful forest species model for genetic and ecological research. In recent years, © 2015 Chen and Cao. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen and Cao BMC Genomics (2015) 16:696 Page 2 of 13 studies on Populus miRNAs have increased [19–21], particularly the expression of miRNAs and the function of the targets in response to abiotic stresses, including cold, heat, drought, flooding, high salt and mechanical stress [22–29]. A larger number of serious diseases occur in poplar, particularly foliar rust disease induced through infections with the rust fungus Melampsora spp., which markedly affects the growth of seeding and saplings. However, few studies have focused on miRNA expression profiling in the response of poplars to biotic stress, particularly pathogen stress [30, 31]. To understand the roles of miRNAs in the response of poplars to pathogenic fungi, we constructed three libraries from uninfected P. szechuanica leaves (control) and leaves infected with avirulent and virulent isolates of M. larici-populina, detected the expression of miRNAs through high-throughput sequencing and analysed the target genes. QRT-PCR was used to analyse the expression of ten miRNAs in plants inoculated for different times. The results of the present study help elucidate the regulatory mechanisms of Populus in response to foliar rust fungal infections. Results Analysis of miRNA sequences Using Solexa sequencing, we constructed three libraries from uninoculated P. szechuanica leaves (control) and leaves inoculated with avirulent (Sb052) and virulent (Th053) isolates of M. larici-populina. A total of 17,801,357, 19,602,945 and 17,962,927 raw reads were obtained from Sb052, Th053 and control libraries, respectively (Table 1). After eliminating low-quality reads and impurities, 17,754,456, 19,544,426 and 17,918,326 high-quality reads were obtained from the three libraries. After discarding 3’ adapter deletions, insertion deletions, 5’ adapter contaminants, poly-A sequences and sequences less than 18 nt from the high-quality reads, 17,557,485, 19,398,402 and 17,764,475 clean reads were used for further analysis. The proportions of clean reads were 98.89, 99.25 and 99.14 % of the total reads obtained from the three libraries, respectively. The small RNA (sRNA) reads were typically 19 to 25 nt in length (Fig. 1). Among these sequences, 21 nt sRNAs were the most abundant in the three libraries, accounting for 57.15 (Control), 52.55 (Sb052) and 48.34 % (Th053) of the total reads, followed by 24 nt sRNAs, which accounted for 17.13, 18.76 and 19.77 % of the total reads, respectively. These results were consistent with previous studies in Populus [24, 25, 27, 28]. However, it was also reported that the most abundant reads length was 24 nt, followed by 21 nt [31, 32], indicating that the miRNAs in Populus are primarily 21 and 24 n (...truncated)