Characterization and Comparative Analysis of Small RNAs in Three Small RNA Libraries of the Brown Planthopper (Nilaparvata lugens)

BACKGROUND: The brown planthopper (BPH), Nilaparvata lugens (Stå;l), which belongs to Homopteran, Delphacidae, is one of the most serious and destructive pests of rice. Feeding BPH with homologous dsRNA in vitro can lead to the death of BPH, which gives a valuable clue to the prevention and control of this pest, however, we know little about its small RNA world. METHODOLOGY/PRINCIPAL FINDINGS: Small RNA libraries for three developmental stages of BPH (CX-male adult, CC-female adult, CY-last instar female nymph) had been constructed and sequenced. It revealed a prolific small RNA world of BPH. We obtained a final list of 452 (CX), 430 (CC), and 381 (CY) conserved microRNAs (miRNAs), respectively, as well as a total of 71 new miRNAs in the three libraries. All the miRNAs had their own expression profiles in the three libraries. The phylogenic evolution of the miRNA families in BPH was consistent with other species. The new miRNA sequences demonstrated some base biases. CONCLUSION: Our study discovered a large number of small RNAs through deep sequencing of three small RNA libraries of BPH. Many animal-conserved miRNA families as well as some novel miRNAs have been detected in our libraries. This is the first achievement to discover the small RNA world of BPH. A lot of new valuable information about BPH small RNAs has been revealed which was helpful for studying insect molecular biology and insect resistant research

Precise and Rapid Validation of Candidate Gene by Allele Specific Knockout With CRISPR/Cas9 in Wild Mice

It is a tempting goal to identify causative genes underlying phenotypic differences among inbred strains of mice, which is a huge reservoir of genetic resources to understand mammalian pathophysiology. In particular, the wild-derived mouse strains harbor enormous genetic variations that have been acquired during evolutionary divergence over 100s of 1000s of years. However, validating the genetic variation in non-classical strains was extremely difficult, until the advent of CRISPR/Cas9 genome editing tools. In this study, we first describe a T cell phenotype in both wild-derived PWD/PhJ parental mice and F1 hybrids, from a cross to C57BL/6 (B6) mice, and we isolate a genetic locus on Chr2, using linkage mapping and chromosome substitution mice. Importantly, we validate the identification of the functional gene controlling this T cell phenotype, Cd44, by allele specific knockout of the PWD copy, leaving the B6 copy completely intact. Our experiments using F1 mice with a dominant phenotype, allowed rapid validation of candidate genes by designing sgRNA PAM sequences that only target the DNA of the PWD genome. We obtained 10 animals derived from B6 eggs fertilized with PWD sperm cells which were subjected to microinjection of CRISPR/Cas9 gene targeting machinery. In the newborns of F1 hybrids, 80% (n = 10) had allele specific knockout of the candidate gene Cd44 of PWD origin, and no mice showed mistargeting of the B6 copy. In the resultant allele-specific knockout F1 mice, we observe full recovery of T cell phenotype. Therefore, our study provided a precise and rapid approach to functionally validate genes that could facilitate gene discovery in classic mouse genetics. More importantly, as we succeeded in genetic manipulation of mice, allele specific knockout could provide the possibility to inactivate disease alleles while keeping the normal allele of the gene intact in human cells

Pairwise comparison results of the expression level of all conserved microRNAs in the three libraries.

<p>Each point represents a miRNA. The X axis and Y axis show the expression level of miRNAs in two libraries, respectively. Red points represent the miRNAs with fold change of expression level between two libraries>2; Blue points represent miRNAs with 1/22; Green points represent miRNAs with fold_change of expression level between two libraries< = 1/2. These conserved miRNAs that had been detected with quantitative PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032860#pone-0032860-g005" target="_blank">Figure 5</a>) were highlighted with other different graph symbols.</p

The clustering results of the conserved miRNAs based on their expression level pairwise comparison results of every two libraries.

<p>Red indicates that the miRNA has higher expression level in treatment sample, green indicates that the miRNA has higher expression in control sample and gray indicates that the miRNA has no expression in at least one sample. (CC2 represents CC; CX2 represents CX; CY2 represents CY). The expression profiles of some conserved miRNAs that had been detected with quantitative PCR (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032860#pone-0032860-g005" target="_blank">Figure 5</a>) were listed out on the right.</p

The pairwise comparisons of total small RNA reads (A, B, C) and unique small RNA reads (D, E, F) in the three libraries.

<p>The overlapping parts were their common sequences. The peculiar parts were their respective specific sequences.</p

Total small RNA type annotations of the three libraries (CC, CY, CX).

<p>Total small RNA type annotations of the three libraries (CC, CY, CX).</p

Total small RNA reads length distributions of the three libraries (CC, CY, and CX).

<p>Total small RNA reads length distributions of the three libraries (CC, CY, and CX).</p

Relative quantitative PCR results of some conserved miRNAs in the three libraries (CC, CX, CY).

<p>Sample libraries were marked as abscissa and the relative expression level of the conserved miRNA as ordinate.</p

Loss of natural resistance to schistosome in T cell deficient rat.

Schistosomiasis is among the major neglected tropical diseases and effective prevention by boosting the immune system is still not available. T cells are key cellular components governing adaptive immune response to various infections. While common laboratory mice, such as C57BL/6, are highly susceptible to schistosomiasis, the SD rats are extremely resistant. However, whether adaptive immunity is necessary for such natural resistance to schistosomiasis in rats remains to be determined. Therefore, it is necessary to establish genetic model deficient in T cells and adaptive immunity on the resistant SD background, and to characterize liver pathology during schistosomiasis. In this study we compared experimental schistosomiasis in highly susceptible C57BL/6 (B6) mice and in resistant SD rats, using cercariae of Schistosoma japonicum. We observed a marked T cell expansion in the spleen of infected B6 mice, but not resistant SD rats. Interestingly, CD3e-/- B6 mice in which T cells are completely absent, the infectious burden of adult worms was significantly higher than that in WT mice, suggesting an anti-parasitic role for T cells in B6 mice during schistosome infection. In further experiments, we established Lck deficient SD rats by using CRISPR/Cas9 in which T cell development was completely abolished. Strikingly, we found that such Lck deficiency in SD rats severely impaired their natural resistance to schistosome infection, and fostered parasite growth. Together with an additional genetic model deficient in T cells, the CD3e-/- SD rats, we confirmed the absence of T cell resulted in loss of natural resistance to schistosome infection, but also mitigated liver immunopathology. Our further experiments showed that regulatory T cell differentiation in infected SD rats was significantly decreased during schistosomiasis, in contrast to significant increase of regulatory T cells in infected B6 mice. These data suggest that T cell mediated immune tolerance facilitates persistent infection in mice but not in SD rats. The demonstration of an important role for T cells in natural resistance of SD rats to schistosomiasis provides experimental evidences supporting the rationale to boost T cell responses in humans to prevent and treat schistosomiasis

The 11 new miRNA members of one miRNA family in BPH.

<p>The 11 new miRNA members of one miRNA family in BPH.</p