scholarly journals Assessing Environmental RNAi in a Non-Model Organism

2019 ◽  
Author(s):  
Mosharrof Mondal ◽  
Jacob Peter ◽  
Obrie Scarbrough ◽  
Alex Flynt
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Spider Mite ◽  
Genetic Manipulation ◽  
Model Organism ◽  
Model Organisms ◽  
Rnai Pathway ◽  
Rnai Technology ◽  
Two Spotted Spider Mite ◽  
Multicellular Organisms

ABSTRACTRNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation in invertebrates and has applications that includes control of pests. Successful RNAi technologies are dependent on delivery method. The most convenient method is likely feeding which is effective in some animals while others are insensitive. Thus, there is a need to develop RNAi technology on a per-species basis, which will require a comprehensive approach for assessing small RNA production from synthetic nucleic acids.Using a biochemical and sequencing approaches we investigated the metabolism of ingested RNAs using the two-spotted spider mite, Tetranychus urticae, as a model for RNAi insensitivity. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. To identify RNAi substrates in T. urticae we characterized processing of synthetic RNAs and those derived from plant transcripts ingested during feeding. Through characterization of read size length and overlaps of small RNA reads, visualization methods were developed that facilitate distinguish trans-acting small RNAs from degradation fragments.Using a strategy that delineates small RNA classes, we found a variety of RNA species are gated into spider mite RNAi pathways, however, potential mature trans-acting RNAs appear very unstable and rare. This suggests spider mite RNAi pathway products that originate as ingested materials may be preferentially metabolized instead of converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers.

scholarly journals Environmental RNAi pathways in the two-spotted spider mite

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Mosharrof Mondal ◽  
Jacob Peter ◽  
Obrie Scarbrough ◽  
Alex Flynt
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Spider Mite ◽  
Genetic Manipulation ◽  
Model Organisms ◽  
Rnai Pathway ◽  
Plant Host ◽  
Rnai Technology ◽  
Two Spotted Spider Mite ◽  
Multicellular Organisms

Abstract Background RNA interference (RNAi) regulates gene expression in most multicellular organisms through binding of small RNA effectors to target transcripts. Exploiting this process is a popular strategy for genetic manipulation and has applications that includes arthropod pest control. RNAi technologies are dependent on delivery method with the most convenient likely being feeding, which is effective in some animals while others are insensitive. The two-spotted spider mite, Tetranychus urticae, is prime candidate for developing RNAi approaches due to frequent occurrence of conventional pesticide resistance. Using a sequencing-based approach, the fate of ingested RNAs was explored to identify features and conditions that affect small RNA biogenesis from external sources to better inform RNAi design. Results Biochemical and sequencing approaches in conjunction with extensive computational assessment were used to evaluate metabolism of ingested RNAs in T. urticae. This chelicerae arthropod shows only modest response to oral RNAi and has biogenesis pathways distinct from model organisms. Processing of synthetic and plant host RNAs ingested during feeding were evaluated to identify active substrates for spider mite RNAi pathways. Through cataloging characteristics of biochemically purified RNA from these sources, trans-acting small RNAs could be distinguished from degradation fragments and their origins documented. Conclusions Using a strategy that delineates small RNA processing, we found many transcripts have the potential to enter spider mite RNAi pathways, however, trans-acting RNAs appear very unstable and rare. This suggests potential RNAi pathway substrates from ingested materials are mostly degraded and infrequently converted into regulators of gene expression. Spider mites infest a variety of plants, and it would be maladaptive to generate diverse gene regulators from dietary RNAs. This study provides a framework for assessing RNAi technology in organisms where genetic and biochemical tools are absent and benefit rationale design of RNAi triggers for T.urticae.

scholarly journals Computational approaches for the analysis of epigenome and transcriptome characterisation in Paramecium tetraurelia

2021 ◽  
Author(s):  
◽  
Sivarajan Karunanithi
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Gc Content ◽  
Model Organisms ◽  
Rnai Pathway ◽  
Paramecium Tetraurelia ◽  
Gene Length ◽  
Histone Marks ◽  
In Cis ◽  
Regulatory Landscape

In the last two decades, our understanding of human gene regulation has improved tremendously. There are plentiful computational methods which focus on integrative data analysis of humans, and model organisms, like mouse and drosophila. However, these tools are not directly employable by researchers working on non-model organisms to answer fundamental biological, and evolutionary questions. We aimed to develop new tools, and adapt existing software for the analysis of transcriptomic and epigenomic data of one such non-model organism, Paramecium tetraurelia, an unicellular eukaryote. Paramecium contains two diploid (2n) germline micronuclei (MIC) and a polyploid (800n) somatic macronuclei (MAC). The transcriptomic and epigenomic regulatory landscape of the MAC genome, which has 80% protein-coding genes and short intergenic regions, is poorly understood. We developed a generic automated eukaryotic short interfering RNA (siRNA) analysis tool, called RAPID. Our tool captures diverse siRNA characteristics from small RNA sequencing data and provides easily navigable visualisations. We also introduced a normalisation technique to facilitate comparison of multiple siRNA-based gene knockdown studies. Further, we developed a pipeline to characterise novel genome-wide endogenous short interfering RNAs (endo-siRNAs). In contrary to many organisms, we found that the endo-siRNAs are not acting in cis, to silence their parent mRNA. We also predicted phasing of siRNAs, which are regulated by the RNA interference (RNAi) pathway. Further, using RAPID, we investigated the aberrations of endo-siRNAs, and their respective transcriptomic alterations caused by an RNAi pathway triggered by feeding small RNAs against a target gene. We find that the small RNA transcriptome is altered, even if a gene unrelated to RNAi pathway is targeted. This is important in the context of investigations of genetically modified organisms (GMOs). We suggest that future studies need to distinguish transcriptomic changes caused by RNAi inducing techniques and actual regulatory changes. Subsequently, we adapted existing epigenomics analysis tools to conduct the first comprehensive epigenomic characterisation of nucleosome positioning and histone modifications of the Paramecium MAC. We identified well positioned nucleosomes shifted downstream of the transcription start site. GC content seems to dictate, in cis, the positioning of nucleosomes, histone marks (H3K4me3, H3K9ac, and H3K27me3), and Pol II in the AT-rich Paramecium genome. We employed a chromatin state segmentation approach, on nucleosomes and histone marks, which revealed genes with active, repressive, and bivalent chromatin states. Further, we constructed a regulatory association network of all the aforementioned data, using the sparse partial correlation network technique. Our analysis revealed subsets of genes, whose expression is positively associated with H3K27me3, different to the otherwise reported negative association with gene expression in many other organisms. Further, we developed a Random Forests classifier to predict gene expression using genic (gene length, intron frequency, etc.) and epigenetic features. Our model has a test performance (PR-AUC) of 0.83. Upon evaluating different feature sets, we found that genic features are as predictive, of gene expression, as the epigenetic features. We used Shapley local feature explanation values, to suggest that high H3K4me3, high intron frequency, low gene length, high sRNA, and high GC content are the most important elements for determining gene expression status. In this thesis, we developed novel tools, and employed several bioinformatics and machine learning methods to characterise the regulatory landscape of the Paramecium’s (epi)genome.

scholarly journals Environmental RNA interference in two-spotted spider mite, Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Nicolas Bensoussan ◽  
Sameer Dixit ◽  
Midori Tabara ◽  
David Letwin ◽  
Maja Milojevic ◽  
...  
Keyword(s):  
Rna Interference ◽  
Tetranychus Urticae ◽  
Pest Control ◽  
Spider Mite ◽  
26S Proteasome ◽  
Rnai Pathway ◽  
Rnai Machinery ◽  
Two Spotted Spider Mite ◽  
Species Specific

Abstract 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.

scholarly journals Environmental RNA interference in two-spotted spider mite, Tetranychus urticae, reveals dsRNA processing requirements for efficient RNAi response

2020 ◽  
Author(s):  
Nicolas Bensoussan ◽  
Sameer Dixit ◽  
Midori Tabara ◽  
David Letwin ◽  
Maja Milojevic ◽  
...  
Keyword(s):  
Rna Interference ◽  
Tetranychus Urticae ◽  
Pest Control ◽  
Spider Mite ◽  
26S Proteasome ◽  
Rnai Pathway ◽  
Rnai Machinery ◽  
Two Spotted Spider Mite ◽  
Species Specific

AbstractComprehensive 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 1,100 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.

Zebrafish as an animal model for the antiviral RNA interference pathway

2021 ◽  
Author(s):  
Yanxin Ren ◽  
Xueyu Li ◽  
Zhonghui Tian ◽  
Yan Xu ◽  
Ruilin Zhang ◽  
...  
Keyword(s):  
Rna Interference ◽  
Sindbis Virus ◽  
Rna Virus ◽  
Model Organism ◽  
Model Organisms ◽  
Rnai Pathway ◽  
Small Interfering Rnas ◽  
Argonaute 2 ◽  
Larval Zebrafish ◽  
Vertebrate Model

The zebrafish (Danio rerio) possesses evolutionarily conserved innate and adaptive immunity as a mammal and has recently become a popular vertebrate model to exploit infection and immunity. Antiviral RNA interference (RNAi) has been illuminated in various model organisms, including Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans and mice. However, to date, there is no report on the antiviral RNAi pathway of zebrafish. Here, we have evaluated the possible use of zebrafish to study antiviral RNAi with Sindbis virus (SINV), vesicular stomatitis virus (VSV) and Nodamura virus (NoV). We find that SINVs and NoVs induce the production of virus-derived small interfering RNAs (vsiRNAs), the hallmark of antiviral RNAi, with a preference for a length of 22 nucleotides, after infection of larval zebrafish. Meanwhile, the suppressor of RNAi (VSR) protein, NoV B2, may affect the accumulation of the NoV in zebrafish. Furthermore, taking advantage of the fact that zebrafish argonaute-2 (Ago2) protein is naturally deficient in cleavage compared with that of mammals, we provide evidence that the slicing activity of human Ago2 can virtually inhibit the accumulation of RNA virus after being ectopically expressed in larval zebrafish. Thus, zebrafish may be a unique model organism to study the antiviral RNAi pathway.

Physiological and molecular mechanisms of salt and water homeostasis in the nematode Caenorhabditis elegans

2013 ◽  
Vol 305 (3) ◽  
pp. R175-R186 ◽  
Author(s):  
Keith P. Choe
Keyword(s):  
Caenorhabditis Elegans ◽  
Regulatory Networks ◽  
Molecular Mechanisms ◽  
Genetic Manipulation ◽  
Model Organisms ◽  
Nematode Caenorhabditis Elegans ◽  
Water Homeostasis ◽  
Hypertonic Stress ◽  
Genetic Mechanisms ◽  
Multicellular Organisms

Intracellular salt and water homeostasis is essential for all cellular life. Extracellular salt and water homeostasis is also important for multicellular organisms. Many fundamental mechanisms of compensation for osmotic perturbations are well defined and conserved. Alternatively, molecular mechanisms of detecting salt and water imbalances and regulating compensatory responses are generally poorly defined for animals. Throughout the last century, researchers studying vertebrates and vertebrate cells made critical contributions to our understanding of osmoregulation, especially mechanisms of salt and water transport and organic osmolyte accumulation. Researchers have more recently started using invertebrate model organisms with defined genomes and well-established methods of genetic manipulation to begin defining the genes and integrated regulatory networks that respond to osmotic stress. The nematode Caenorhabditis elegans is well suited to these studies. Here, I introduce osmoregulatory mechanisms in this model, discuss experimental advantages and limitations, and review important findings. Key discoveries include defining genetic mechanisms of osmolarity sensing in neurons, identifying protein damage as a sensor and principle determinant of hypertonic stress resistance, and identification of a putative sensor for hypertonic stress associated with the extracellular matrix. Many of these processes and pathways are conserved and, therefore, provide new insights into salt and water homeostasis in other animals, including mammals.

scholarly journals Comparative genome and transcriptome analyses reveal innate differences in response to host plants by two color forms of the two-spotted spider mite Tetranychus urticae

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Shi-Mei Huo ◽  
Zhi-Chao Yan ◽  
Feng Zhang ◽  
Lei Chen ◽  
Jing-Tao Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tetranychus Urticae ◽  
Host Plants ◽  
Spider Mite ◽  
Principal Component ◽  
Consistent Difference ◽  
Plant Interactions ◽  
Specific Expression ◽  
Two Spotted Spider Mite ◽  
Biological Performance

Abstract Background The two-spotted spider mite, Tetranychus urticae, is a major agricultural pest with a cosmopolitan distribution, and its polyphagous habits provide a model for investigating herbivore-plant interactions. There are two body color forms of T. urticae with a different host preference. Comparative genomics and transcriptomics are used here to investigate differences in responses of the forms to host plants at the molecular level. Biological responses of the two forms sourced from multiple populations are also presented. Results We carried out principal component analysis of transcription changes in three red and three green T. urticae populations feeding on their original host (common bean), and three hosts to which they were transferred: cotton, cucumber and eggplant. There were differences among the forms in gene expression regardless of their host plant. In addition, different changes in gene expression were evident in the two forms when responding to the same host transfer. We further compared biological performance among populations of the two forms after feeding on each of the four hosts. Fecundity of 2-day-old adult females showed a consistent difference between the forms after feeding on bean. We produced a 90.1-Mb genome of the red form of T. urticae with scaffold N50 of 12.78 Mb. Transcriptional profiles of genes associated with saliva, digestion and detoxification showed form-dependent responses to the same host and these genes also showed host-specific expression effects. Conclusions Our research revealed that forms of T. urticae differ in host-determined transcription responses and that there is form-dependent plasticity in the transcriptomic responses. These differences may facilitate the extreme polyphagy shown by spider mites, although fitness differences on hosts are also influenced by population differences unrelated to color form.

Individuality, Organisms, and Cell Differentiation

2018 ◽  
Author(s):  
Melinda Bonnie Fagan
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Cells ◽  
Functional Integration ◽  
Model Organism ◽  
Model Organisms ◽  
Strong Version ◽  
Mammalian Development ◽  
Multicellular Organisms ◽  
Early Mammalian Development

This chapter builds on earlier arguments concerning the individuality of stem cells. The author has argued in previous work that stem cells are not biological individuals in the same way as specialized cells of multicellular organisms (e.g., neurons, red blood cells, muscle cells) but that some stem cells (cultured pluripotent stem cells) can be considered biological individuals by analogy with multicellular organisms. More precisely, the author claims that cultured pluripotent stem cells can be considered model organisms for studying early mammalian development. An important objection to this model organism thesis is that cultured pluripotent stem cells lack the organization (functional integration and cohesive unity) required for an entity to be an organism. This chapter explicates and rebuts a strong version of this objection and, in the process, clarifies the ontology of stem cells as experimental entities.

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