Re: Spider Man 2 Pc Requirements
Osoulo Lejeune
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Comprehensive understanding of pleiotropic roles of RNAi machinery highlighted the conserved chromosomal functions of RNA interference. The consequences of the evolutionary variation in the core RNAi pathway genes are mostly unknown, but may lead to the species-specific functions associated with gene silencing. The two-spotted spider mite, Tetranychus urticae, is a major polyphagous chelicerate pest capable of feeding on over 1100 plant species and developing resistance to pesticides used for its control. A well annotated genome, susceptibility to RNAi and economic importance, make T. urticae an excellent candidate for development of an RNAi protocol that enables high-throughput genetic screens and RNAi-based pest control. Here, we show that the length of the exogenous dsRNA critically determines its processivity and ability to induce RNAi in vivo. A combination of the long dsRNAs and the use of dye to trace the ingestion of dsRNA enabled the identification of genes involved in membrane transport and 26S proteasome degradation as sensitive RNAi targets. Our data demonstrate that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the components of the RNAi machinery make T. urticae a potent experimental system to study the evolution of RNAi pathways.
The ability of double-stranded RNA (dsRNA) to inhibit the expression of a complementary target gene in a process named RNA interference (RNAi) was first demonstrated in plants and Caenorhabditis elegans1,2 and was since discovered in a wide range of organisms3,4,5. The RNAi is guided by small RNAs that can be generated from different precursor RNA molecules. Short interfering RNAs (siRNAs) are generated from dsRNAs. They interfere with the expression of viral and transposon-originating transcripts or direct chromatin modifications that are essential for proper chromosomal functions6,7. Micro-RNAs (miRNAs) are processed from genome-encoded hairpins that contain local stem-loop structures. These precursor RNAs undergo a series of maturation steps to generate Argonaute-associated miRNAs that primarily silence protein-coding mRNAs through either translational inhibition or mRNA degradation. Finally, single-stranded RNA (ssRNA) precursors, transcribed from chromosomal loci that mostly consist of remnants of transposable element sequences, give rise to PIWI-interacting RNAs (piRNAs). They are mainly active in germ-line tissues, where they transcriptionally silence transposable elements7.
Understanding of the siRNA RNAi pathway led to the use of the exogenously supplied dsRNA to trigger RNAi (termed environmental RNAi8). This opened possibilities to develop RNAi as a reverse genetics tool9,10,11,12,13 and more recently as an environmentally friendly strategy for pest control14,15. The stability of the exogenous dsRNAs in body fluids, their cellular uptake, and spread to distal tissues that were not in direct contact with the initially delivered dsRNA were identified as key elements for the efficient environmental RNAi. However, the variability in the gut pH and the presence of dsRNases in gut and/or hemolymph that lead to dsRNA hydrolysis and degradation, limit the use of RNAi in many insect clades (reviewed in Cooper et al.16). Consequently, many arthropods (e.g. some lepidopterans) are refractory to the environmental RNAi. Others (such as some coleopterans, blattodeans and orthopterans) can induce the environmental RNAi only by the injection of dsRNA, restricting its use to reverse genetics. Finally, only a subset of arthropods that respond to the orally delivered dsRNAs are potential candidate species for whom environmental RNAi can be used as a reverse genetics and a pest control tool16.
The two-spotted spider mite, Tetranychus urticae, is an important agricultural pest that feeds on more than a thousand plant species including over 150 crops17. Consistent with its outstanding xenobiotic resistance against plant allelochemicals, T. urticae populations can rapidly develop resistance to pesticides used for its control18, hindering the control of mite infestations in agricultural settings. T. urticae is also a model chelicerate whose genome is well annotated and constantly updated19. In addition, numerous datasets describing mite transcriptional changes associated with developmental progression, pesticide resistance, or upon host-shift, are available19,20,21,22,23,24. Furthermore, the candidate loci underlying mite physiological processes (e.g. diapause, carotenoid biosynthesis) and pesticide resistance using forward genetic approaches have been recently identified25,26. However, the assessment of the in vivo function of candidate loci, at the level of an otherwise unperturbed whole organism, is so far lacking. In the absence of well-established reverse genetics tools, a demonstration that proteins encoded by candidate loci have capabilities to modify mite physiology rely on the expression of candidate genes in heterologous systems or on in vitro assays. Thus, the development of reverse genetics tools is essential for the understanding of gene function and the unique features of T. urticae biology.
The analysis of the T. urticae genome identified significant variation in RNAi core machinery compared with other arthropods19. T. urticae harbours a single copy of pasha and drosha genes, two dicer homologs, seven argonaute and seven piwi genes. T. urticae lacks R2D2 that is a co-factor of Dicer-2 in Drosophila, however, it contains two copies of loquacious gene. Finally, unlike in insects and crustaceans, the mite genome contains five homologs of RNA dependent RNA polymerase (RdRP) genes19. Pleiotropic roles of RNAi in gene silencing and chromosomal functions (reviewed in Gutbrod and Martienssen6), together with the contribution of core RNAi proteins to more than one RNAi pathway, confines an understanding of the functional implications of the diversity and evolutionary variation in RNAi machinery7,16,27,28. Regardless, it has been demonstrated that maternal injections of dsRNA and siRNA, and oral delivery of dsRNA can induce environmental RNAi in T. urticae29,30,31,32. Here, we investigated the effect of sequence composition and length of the exogenous dsRNA on the efficiency of RNAi and its processivity by Dicer using both in vivo and in vitro analysis. Understanding the length requirement of the exogenous dsRNA and utilization of tracer dye to control the variability in dsRNA ingestion led to the development of a highly efficient RNAi protocol. The protocol was rigorously tested against twelve T. urticae homologs of Tribolium castaneum sensitive RNAi targets33, demonstrating that environmental RNAi can be an efficient reverse genetics and pest control tool in T. urticae. In addition, the species-specific properties together with the variation in the complement of RNAi pathway genes identified in the T. urticae genome make the two-spotted spider mite a potent experimental system to study the evolution of RNAi pathways.
Even the length-optimized dsRNAs induced RNAi only in a subset of treated mites, Fig. 2. A variation in dsRNA uptake, tracked by the addition of dye into dsRNA solution, was previously reported for aphids39. To test if mites soaked in dsRNA solution ingest dsRNA differentially, we used a 6% blue food dye that can be easily visualized in mite posterior midgut, Fig. 3A, and mixed it in dsRNA solution. Upon soaking, 64% and 66% of the total mites treated with dsRNA-TuVATPase-B and dsRNA-TuCOPB2-B showed blue color in the posterior midgut, respectively. When mites were preselected for the presence of blue dye in their gut, the proportion of mites displaying the RNAi-associated phenotypes was greater than 90% (94% and 95% of mites treated with dsRNA-TuVATPase-B and dsRNA-TuCOPB2-B displayed dark-body and spotless phenotypes, respectively (Fig. 3B)). Mites without the visible accumulation of dye had a significantly lower frequency of body phenotypes associated with RNAi responses (Fig. 3B). Thus, the blue dye could be used as a dsRNA tracer, securing the RNAi responsiveness in the majority of treated mites.
Several application methods for the oral delivery of dsRNA to T. urticae have been developed30,32,38. These methods use a wide range of dsRNA concentrations, from 40 up to 1 g/L. To determine the effective dsRNA concentrations and the potential toxicity of high concentrations of dsRNAs due to the possible oversaturation of the RNAi machinery, we applied dsRNA-TuVATPase, dsRNA-TuCOPB2 and dsRNA-NC in the range of 20 to 1280 ng/L and scored mite survivorship, Fig. 4. At 20 ng/L, individual dsRNAs against TuVATPase and TuCOPB2 did not affect mite mortality, Fig. 4A. However, the mixture of these dsRNAs (at the concentration of 20 ng/L each) acted synergistically and resulted in a significant decrease of mite survivorship, Fig. 4B. dsRNA-TuVATPase and dsRNA-TuCOPB2 induced significant mite mortality (80%) at 40 and 80 ng/L, respectively, and their responses were saturated at 160 ng/L, Fig. 4A. Therefore, even though the effective concentrations of dsRNA may vary dependent on the target, the concentration of 160 ng/L of dsRNA appears to be sufficient to induce the full RNAi response and was used in subsequent experiments. High concentrations of dsRNAs, at 1280 ng/L, did not affect mite mortality (Fig. 4C), indicating that dsRNA at this concentration was not toxic to mites and did not oversaturate the RNAi machinery.
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