A Yeast RNA-Interference Pesticide Targeting the Irx Gene Functions as a Broad-Based Mosquito Larvicide and Adulticide

Concerns for widespread insecticide resistance and the unintended impacts of insecticides on nontarget organisms have generated a pressing need for mosquito control innovations. A yeast RNAi-based insecticide that targets a conserved site in mosquito Irx family genes, but which has not yet been identified in the genomes of nontarget organisms, was developed and characterized. Saccharomyces cerevisiae constructed to express short hairpin RNA (shRNA) matching the target site induced significant Aedes aegypti larval death in both lab trials and outdoor semi-field evaluations. The yeast also induced high levels of mortality in adult females, which readily consumed yeast incorporated into an attractive targeted sugar bait (ATSB) during simulated field trials. A conserved requirement for Irx function as a regulator of proneural gene expression was observed in the mosquito brain, suggesting a possible mode of action. The larvicidal and adulticidal properties of the yeast were also verified in Aedes albopictus, Anopheles gambiae, and Culexquinquefasciatus mosquitoes, but the yeast larvicide was not toxic to other nontarget arthropods. These results indicate that further development and evaluation of this technology as an ecofriendly control intervention is warranted, and that ATSBs, an emerging mosquito control paradigm, could potentially be enriched through the use of yeast-based RNAi technology.

Development of a Viral RdRp-Assisted Gene Silencing System and Its Application in the Identification of Host Factors of Plant (+)RNA Virus

Gene silencing induced by hairpin RNA or virus infection expression is one of the major tools in genetics studies in plants. However, when dealing with essential genes, virus-induced gene silencing (VIGS) and transgenic expression of hairpin RNA could lead to plant death, while transient expression of hairpin RNA in leaves is often less competent in downregulating target gene mRNA levels. Here, we developed a transient double-stranded RNA (dsRNA) expression system assisted by a modified viral RNA-dependent RNA polymerase (RdRp) in plant leaves. We show that this system is more effective in inducing gene silencing than the intron-spliced hairpin RNA expression. Furthermore, by using this system, we tested the role of the early secretory pathway during infection of Soybean mosaic potyvirus (SMV). We found that key components of the coat protein complex II vesicles are required for the multiplication of SMV. Overall, this dsRNA-based gene silencing system is effective in downregulating plant gene expression and can be used to identify host genes involved in plant-virus interactions.

Intron-Containing Hairpin RNA Interference Vector for OBP8 Show Promising Mortality in Peach Potato Aphid

Myzus persicae is a devastating pest affecting potato production. RNA Interference technology is used against essential odorant binding protein 8 (OBP8) to enhance protection against Myzus persicae in potato. Gene was isolated, sequenced and GenBank IDs were allotted and ERNAi was used to design siRNA targets from OBP8 with no off-targets. Multiple Sequence Alignment show M. persicae OBP8 resemblance with Acyrthosiphon pisum, Rhopalosiphum maidis, Aphis fabae, and Sitobion avenae. DsRNA (7 µg/µl) oral acquisition had resulted in 69% mortality and 58% reduction in OBP8 expression 8D post dsRNA feeding in comparison to control. Golden Gate (GG) cloning based RNAigg is used for RNA interference taking advantage of type IIs restriction enzyme Eco31I. Agro infiltration assay was used for introduction of intron-containing hairpin RNA (ihpRNA) in Solanum tuberosum. Aphids feeding on transgenic S. tuberosum show 57.6% mortality and 49% reduction in OBP8 expression 8d post-feeding in comparison to control. This work proves OBP8 as promising ihpRNA targets in potato and related crops for whom Myzus is a devastating target.

Total Flavones Isolated from Lycium Barbarum L. Inhibit the Proliferation, Migration and Invasion of GlioblastomaU-87MG Cells by Decreasing Formyl Peptide Receptor 1 Expression

Background: To study the role and mechanisms of total flavones from Lycium barbarum L. (TFL) and FPR1 in the growth of glioblastoma U-87MG cells.Main Methods: CCK-8, wound-healing and Transwell were used for investigating proliferation, motility and invasion of U87 cells after treating with total flavones. RT-qPCR and Western blot were used to study the effect of total flavones on proliferating cell nuclear antigen (PCNA), matrix metalloproteinase2 (MMP2) and FPR1. The short hairpin RNA and FPR agonist fMLP were used to delineate the role of FPR1.Results: TFL was successfully isolated, and its concentration was determined to be 6.205 mg/l. TFL inhibited the proliferation, migration and invasion of U-87MG cells in a time and dose-dependent manner compared to controls. Decreasing FPR1 expression using short hairpin RNA significantly inhibited the migration and invasion of U-87MG cells. Notably, increased expression of FPR1 and treatment with FPR-agonist peptides such as N-formylmethionyl-leucyl-phenylalanine induced the migration and invasion of U-87MG cells, which was significantly decreased when the cells were treated with TFL. Conclusion: TFL inhibits the proliferation, migration and invasion of human glioblastoma U-87MG cells through decreasing the expression of FPR1. These findings provide valuable evidence for the development of antitumor drugs.

Enhanced RNAi stability through imperfect inverted repeats: nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes in plants

Hairpin RNA (hpRNA) transgenes, with a perfect inverted-repeat (IR) DNA, have been the most successful RNA interference (RNAi) method in plants. Here we show that hpRNA transgenes were invariably methylated in the IR DNA and the adjacent promoter, causing transcriptional self-silencing and preventing the full potential of RNAi. Nucleotide substitutions in the sense sequence, which disrupts the perfect IR DNA structure, were sufficient to prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine (C) with thymine (T) nucleotides, in a G:U hpRNA design, prevented DNA methylation and self-silencing but still allowed for the formation of perfect hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90–96% of transgenic lines, compared to 57–65% for the traditional hpRNA design. Furthermore, while a traditional hpRNA transgene showed increasing DNA methylation and self-silencing from cotyledons to true leaves, the G:U transgenes avoided this developmental progression of self-silencing and induced RNAi throughout plant growth. The G:U and traditional hpRNA transgenes generated small interfering RNA (siRNA) with different 5’ phosphorylation, which resembled the endogenous tasiRNA and miRNA, respectively. Furthermore, our results suggest that siRNAs from the two transgene designs function differently to induce target DNA methylation, one (from traditional hpRNA) through the canonical RdDM pathway and the other (G:U hpRNA) a non-canonical pathway. Our study not only revealed a methylation-resistant RNAi transgene design but also provided new mechanistic insights into small RNA biogenesis and function in plants.