Identification of microRNAs from Plutella xylostella larvae associated with parasitization by Diadegma semiclausum

MicroRNAs (miRNAs) as small non-coding RNAs play important roles in many biological processes such as development, cell signaling and immune response. Small RNA deep sequencing technology provided an opportunity for a thorough survey of miRNAs in a global key pest Plutella xylostella as well as comparative analysis of miRNA expression profile of the insect in association with parasitization by Diadegma semiclausum. Combining the deep sequencing data and bioinformatics, 235 miRNAs were identified from P. xylostella. Differential expression of host cellular miRNAs in response to parasitism was examined by making small RNA libraries from parasitized and naive second instar larvae of P. xylostella. Bantam, miR-276*, miR-10, miR-31 and miR-184 were detected as five most abundant miRNAs in both libraries and 96 miRNAs were identified that were differentially expressed after parasitization. Bantam*, miR-184 and miR-281* were significantly down-regulated and two miRNAs miR-279b and miR-2944b* were highly induced in parasitized larvae. Interestingly, high copy numbers and differential expression of several miRNA passenger strands (miRNA*) suggest their potential roles in host-parasitoid interaction. In conclusion, expression profiling of miRNAs provided insights into their possible involvement in insect immune response to parasitism and offer an important resource for further studies.     

Identification and Characterization of MicroRNAs in Pearl Oyster Pinctada martensii by Solexa Deep Sequencing

MicroRNAs (miRNAs) are short-nucleotide RNA molecules that function as negative regulators of gene expression in various organisms. However, miRNAs of Pinctada martensii have not been reported yet. P. martensii is one of the main species cultured for marine pearl production in China and Japan. In order to obtain the repertoire of miRNAs in P. martensii, we constructed and sequenced small RNA libraries prepared from P. martensii by Solexa deep sequencing technology and got a total of 27,479,838 reads representing 3,176,630 distinct sequences. After removing tRNAs, rRNAs, snRNAs, and snoRNAs, 10,596,306 miRNA reads representing 18,050 distinct miRNA reads were obtained. Based on sequence similarity and hairpin structure prediction, 258 P. martensii miRNAs (pm-miRNA) were identified. Among these pm-miRNAs, 205 were conserved across the species, whereas 53 were specific for P. martensii. The 3′ end sequence of U6 snRNA was obtained from P. martensii by 3′ rapid amplification of cDNA end PCR reaction and sequence-directed cloning. Eight conserved pm-miRNAs and two novel pm-miRNAs were validated by stem-loop quantitative real-time PCR with U6 snRNA as an internal reference gene. pm-miRNAs and the reported biomineralization-related genes were subjected to target analysis by using target prediction tools. Some of the pm-miRNAs, such as miR-2305 and miR-0046, were predicted to participate in biomineralization by regulating the biomineralization-related genes. Thus, this study demonstrated a large-scale characterization of pm-miRNAs and their potential function in biomineralization, providing a foundation to understand shell formation.     

Genome-wide profiling of novel and conserved <Emphasis Type="Italic">Populus</Emphasis> microRNAs involved in pathogen stress response by deep sequencing

MicroRNAs (miRNAs) are small RNAs, generally of 20–23 nt, that down-regulate target gene expression during development, differentiation, growth, and metabolism. In Populus, extensive studies of miRNAs involved in cold, heat, dehydration, salinity, and mechanical stresses have been performed; however, there are few reports profiling the miRNA expression patterns during pathogen stress. We obtained almost 38 million raw reads through Solexa sequencing of two libraries from Populus inoculated and uninoculated with canker disease pathogen. Sequence analyses identified 74 conserved miRNA sequences belonging to 37 miRNA families from 154 loci in the Populus genome and 27 novel miRNA sequences from 35 loci, including their complementary miRNA* strands. Intriguingly, the miRNA* of three conserved miRNAs were more abundant than their corresponding miRNAs. The overall expression levels of conserved miRNAs increased when subjected to pathogen stress, and expression levels of 33 miRNA sequences markedly changed. The expression trends determined by sequencing and by qRT-PCR were similar. Finally, nine target genes for three conserved miRNAs and 63 target genes for novel miRNAs were predicted using computational analysis, and their functions were annotated. Deep sequencing provides an opportunity to identify pathogen-regulated miRNAs in trees, which will help in understanding the regulatory mechanisms of plant defense responses during pathogen infection.     

Identification and comparative analysis of the pearl oyster <Emphasis Type="Italic">Pinctada fucata</Emphasis> hemocytes microRNAs in response to <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> infection

MicroRNAs (miRNAs) are a class of noncoding RNA molecules that function as negative regulators of gene expression and play important roles in a wide spectrum of biological processes, including in immune response. However, the physiological regulation function of Pinctada fucata miRNAs, specially their immunomodulation has not been explored yet. Here, two small RNA libraries from hemocytes of P. fucata with or without Vibrio alginolyticus infection were constructed and sequenced using the high-throughput Illumina deep sequencing technology. In total, 11,939,992 and 11,083,327 raw reads, corresponding to 10,993,546 and 9,988,179 clean reads, were respectively obtained in the control and infected libraries. A total of 276 miRNAs, including 225 known miRNAs and 51 putative novel miRNAs, were identified by bioinformatic analysis. By using pairwise comparison between two libraries, 93 miRNAs were found to be significantly differentially expressed, with 42 and 51 miRNAs exhibiting up-regulation and down-regulation, respectively. Thereinto, some known miRNAs were considered to be immune-related. Real-time PCR were implemented for 6 miRNAs co-expressed in the control and infected samples, and agreement was confirmed between the high-throughput sequencing and real-time PCR data. After miRNA targets were predicted, GO and KEGG pathway enrichment analysis were performed, and the results indicated that ten of the differentially expressed miRNAs were involved in immune-related pathways, and might participate in the host immune response to V. alginolyticus. These results of identification and comparative analysis of miRNAs might deepen our understanding of host-pathogen interactions and immune defense mechanisms in P. fucata.     

Conserved and novel miRNAs in the legume <Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> in response to stress

MicroRNAs (miRNAs) are small RNA molecules recognized as important regulators of gene expression. Although plant miRNAs have been extensively studied in model systems, less is known in other plants with limited genome sequence data. We are interested in the identification of miRNAs in Phaseolus vulgaris (common bean) to uncover different plant strategies to cope with adverse conditions and because of its relevance as a crop in developing countries. Here we present the identification of conserved and candidate novel miRNAs in P. vulgaris present in different organs and growth conditions, including drought, abscisic acid treatment, and Rhizobium infection. We also identified cDNA sequences in public databases that represent the corresponding miRNA precursors. In addition, we predicted and validated target mRNAs amongst reported EST and cDNAs for P. vulgaris. We propose that the novel miRNAs present in common bean and other legumes, are involved in regulation of legume-specific processes including adaptation to diverse external cues. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and comparative analysis of the Eriocheir sinensis microRNA transcriptome response to Spiroplasma eriocheiris infection using a deep sequencing approach

The Chinese mitten crab Eriocheir sinensis is one of the most important freshwater aquaculture crustacean species in China. MicroRNAs (miRNAs) are small non-coding RNAs that are important effectors in the intricate host-pathogen interaction network. To increase the repertoire of miRNAs characterized in crustaceans and to examine the relationship between host miRNA expression and pathogen infection, we used the Illumina/Solexa deep sequencing technology to sequence two small RNA libraries prepared from haemocytes of E. sinensis under normal conditions and during infection with Spiroplasma eriocheiris. The high-throughput sequencing resulted in approximately 30,975,151 and 30,826,277 raw reads corresponding to 12,077,088 and 16,271,545 high-quality mappable reads for the normal and infected haemocyte samples, respectively. Bioinformatic analyses identified 735 unique miRNAs, including 36 that are conserved in crustaceans, 134 that are novel to crabs but are present in other arthropods (PN-type), and 565 that are completely new (PC-type). Two hundred twenty-eight unique miRNAs displayed significant differential expression between the normal and infected haemocyte samples (p < 0.0001). Of these, 133 (58%) were significantly up-regulated and 95 (42%) were significantly down-regulated upon challenge with S. eriocheiris. Real-time quantitative PCR (RT-qPCR) experiments were preformed for 10 miRNAs of the two samples, and agreement was found between the sequencing and RT-qPCR data. To our knowledge, this is the first report of comprehensive identification of E. sinensis miRNAs and of expression analysis of E. sinensis miRNAs after exposure to S. eriocheiris. Many miRNAs were differentially regulated when exposed to the pathogen, and these findings support the hypothesis that certain miRNAs might be essential in host-pathogen interactions. Our results suggest that elucidation of the molecular mechanisms responsible for miRNA regulation of the host’s innate immune system should help with the development of new control strategies to prevent or treat S. eriocheiris infections in crustaceans.     

Involvement of MicroRNAs in Infection of Silkworm with Bombyx mori Cytoplasmic Polyhedrosis Virus (BmCPV)

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is one of the most important pathogens of silkworm. MicroRNAs (miRNAs) have been demonstrated to play key roles in regulating host-pathogen interaction. However, there are limited reports on the miRNAs expression profiles during insect pathogen challenges. In this study, four small RNA libraries from BmCPV-infected midgut of silkworm at 72 h post-inoculation and 96 h post-inoculation and their corresponding control midguts were constructed and deep sequenced. A total of 316 known miRNAs (including miRNA*) and 90 novel miRNAs were identified. Fifty-eight miRNAs displayed significant differential expression between the infected and normal midgut (P value < = 0.01 and fold change > = 2.0 or < = 0.5), among which ten differentially expressed miRNA were validated by qRT-PCR method. Further bioinformatics analysis of predicted target genes of differentially expressed miRNAs showed that the miRNA targets were involved in stimulus and immune system process in silkworm.