The 'Trojan horse' approach for successful RNA interference in insects.

2021 ◽  
pp. 25-39
Author(s):  
Dimitrios Kontogiannatos ◽  
Anna Kolliopoulou ◽  
Luc Swevers
Keyword(s):  
Rna Interference ◽  
Insect Cells ◽  
New Technologies ◽  
Plant Protection ◽  
Constitutive Expression ◽  
Ease Of Use ◽  
Rnai Efficiency ◽  
Insect Gut ◽  
Species Specific

Abstract Since the discovery of RNA interference in 1998 as a potent molecular tool for the selective downregulation of gene expression in almost all eukaryotes, increasing research is being performed in order to discover applications that are useful for the pharmaceutical and chemical industry. The ease of use of double-stranded RNA for targeted in vivo gene silencing in animal cells and tissues gave birth to a massive interest from industry in order to discover biotechnological applications for human health and plant protection. For insects, RNAi became the 'Holy Grail' of pesticide manufacturing, because this technology is a promising species-specific environmentally friendly approach to killing natural enemies of cultured plants and farmed animals. The general idea to use RNAi as a pest-control agent originated with the realization that dsRNAs that target developmentally or physiologically important insect genes can cause lethal phenotypes as a result of the specific gene downregulation. Most importantly to achieve this, dsRNA is not required to be constitutively expressed via a transgene in the targeted insect but it can be administrated orally after direct spraying on the infested plants. Similarly, dsRNAs can be administered to pests after constitutive expression as a hairpin in plants or bacteria via stable transgenesis. Ideally, this technology could have already been applied in integrated pest management (IPM) if improvements were not essential in order to achieve higher insecticidal effects. There are many limitations that decrease RNAi efficiency in insects, which arise from the biochemical nature of the insect gut as well as from deficiencies in the RNAi core machinery, a common phenomenon mostly observed in lepidopteran species. To overcome these obstacles, new technologies should be assessed to ascertain that the dsRNA will be transferred intact, stable and in high amounts to the targeted insect cells. In this chapter we will review a wide range of recent discoveries that address the delivery issues of dsRNAs in insect cells, with a focus on the most prominent and efficient technologies. We will also review the upcoming and novel use of viral molecular components for the successful and efficient delivery of dsRNA to the insect cell.

The 'Trojan horse' approach for successful RNA interference in insects.

2021 ◽  
pp. 25-39
Author(s):  
Dimitrios Kontogiannatos ◽  
Anna Kolliopoulou ◽  
Luc Swevers
Keyword(s):  
Rna Interference ◽  
Insect Cells ◽  
New Technologies ◽  
Plant Protection ◽  
Constitutive Expression ◽  
Ease Of Use ◽  
Rnai Efficiency ◽  
Insect Gut ◽  
Species Specific

Abstract Since the discovery of RNA interference in 1998 as a potent molecular tool for the selective downregulation of gene expression in almost all eukaryotes, increasing research is being performed in order to discover applications that are useful for the pharmaceutical and chemical industry. The ease of use of double-stranded RNA for targeted in vivo gene silencing in animal cells and tissues gave birth to a massive interest from industry in order to discover biotechnological applications for human health and plant protection. For insects, RNAi became the 'Holy Grail' of pesticide manufacturing, because this technology is a promising species-specific environmentally friendly approach to killing natural enemies of cultured plants and farmed animals. The general idea to use RNAi as a pest-control agent originated with the realization that dsRNAs that target developmentally or physiologically important insect genes can cause lethal phenotypes as a result of the specific gene downregulation. Most importantly to achieve this, dsRNA is not required to be constitutively expressed via a transgene in the targeted insect but it can be administrated orally after direct spraying on the infested plants. Similarly, dsRNAs can be administered to pests after constitutive expression as a hairpin in plants or bacteria via stable transgenesis. Ideally, this technology could have already been applied in integrated pest management (IPM) if improvements were not essential in order to achieve higher insecticidal effects. There are many limitations that decrease RNAi efficiency in insects, which arise from the biochemical nature of the insect gut as well as from deficiencies in the RNAi core machinery, a common phenomenon mostly observed in lepidopteran species. To overcome these obstacles, new technologies should be assessed to ascertain that the dsRNA will be transferred intact, stable and in high amounts to the targeted insect cells. In this chapter we will review a wide range of recent discoveries that address the delivery issues of dsRNAs in insect cells, with a focus on the most prominent and efficient technologies. We will also review the upcoming and novel use of viral molecular components for the successful and efficient delivery of dsRNA to the insect cell.

Mechanisms, Applications, and Challenges of Insect RNA Interference

2020 ◽  
Vol 65 (1) ◽  
pp. 293-311 ◽  
Author(s):  
Kun Yan Zhu ◽  
Subba Reddy Palli
Keyword(s):  
Rna Interference ◽  
Pest Management ◽  
New Technologies ◽  
Insect Pest ◽  
Management Strategies ◽  
Cationic Liposome ◽  
Delivery Methods ◽  
Nontarget Effects ◽  
Development Of Resistance ◽  
Rnai Efficiency

The RNA interference (RNAi) triggered by short/small interfering RNA (siRNA) was discovered in nematodes and found to function in most living organisms. RNAi has been widely used as a research tool to study gene functions and has shown great potential for the development of novel pest management strategies. RNAi is highly efficient and systemic in coleopterans but highly variable or inefficient in many other insects. Differences in double-stranded RNA (dsRNA) degradation, cellular uptake, inter- and intracellular transports, processing of dsRNA to siRNA, and RNA-induced silencing complex formation influence RNAi efficiency. The basic dsRNA delivery methods include microinjection, feeding, and soaking. To improve dsRNA delivery, various new technologies, including cationic liposome–assisted, nanoparticle-enabled, symbiont-mediated, and plant-mediated deliveries, have been developed. Major challenges to widespread use of RNAi in insect pest management include variable RNAi efficiency among insects, lack of reliable dsRNA delivery methods, off-target and nontarget effects, and potential development of resistance in insect populations.

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.

scholarly journals Specifics of pesticides effects on the phytopathogenic bacteria

2016 ◽  
Vol 23 (2) ◽  
pp. 311-331 ◽  
Author(s):  
Volodymyr Patyka ◽  
Natalia Buletsa ◽  
Lidiya Pasichnyk ◽  
Natalia Zhitkevich ◽  
Antonina Kalinichenko ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Plant Protection ◽  
Plant Protection Products ◽  
Ease Of Use ◽  
Chemical Pollution ◽  
Resistance Response ◽  
Phytopathogenic Bacteria ◽  
Long Term Storage

Abstract The data concerning the effects of pesticides of different nature on the phytopathogenic bacteria was examined and summarized. Without extensive research on the mechanisms of interaction between pathogenic bacteria and pesticides in the literature review a similar message about microorganisms of soil and phyllosphere are included. The bacteria can be suppressed permanently by pesticides with a mechanism of action that universally affects biological processes in living systems. Long-term storage, ease of use and fast visible effect are the advantages of synthetic pesticides remedies. But chemical pollution, shifts in the balance of ecosystems, unpredictable effects of chemical pesticides on non-target objects are the drawbacks. Stimulation of resistance response in plants is unifying factor for all types of biopesticides. This is realized through localization of the pathogen during infection, blocking its further penetration, distribution and reproduction. The results of the study of effects of plant protection products on the phytopathogenic bacteria of main crops are described. Among all tested pesticides, thiocarbamate fungicides demonstrated significant inhibitory action on phytopathogens, but their effect may be neutralized by other constituents of multicomponent preparations. Triazole fungicides affect the causative agents of bacterioses of crops at a dose of more than 1% of the active substance in the nutrient medium. Insecticides and herbicides have little or no effect on phytopathogenic bacteria; however they can enhance morphological dissociations of some Pseudomonas strains, thereby increasing their ability to survive. The disadvantage of many biopesticides against phytopathogenic microorganisms is the difference between their efficacy in vitro and in vivo that is why the desired result is not achieved in field condition. In addition, biological pesticides often lose their activity causing the problem of constant search for new active antagonists. The fact that the sensitivity of phytopathogenic bacteria to pesticides is strain-dependent should be considered in practice, particularly, assessment of the antibacterial action of various preparations should not be limited to a single bacterial strain.

scholarly journals Denaturing gradient gel electrophoresis a tool for plant protection research

2003 ◽  
Vol 56 ◽  
pp. 143-150 ◽  
Author(s):  
M. O'Callaghan ◽  
E.M. Gerard ◽  
G.H.J. Heilig ◽  
H. Zhang ◽  
T.A. Jackson ◽  
...  
Keyword(s):  
Microbial Communities ◽  
Gel Electrophoresis ◽  
New Technologies ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Plant Protection ◽  
Microbial Populations ◽  
Significant Challenge ◽  
Community Profiling ◽  
Gradient Gel Electrophoresis ◽  
Insect Gut

Analysis of microbial communities associated with plants insects and soil has been a significant challenge for plant protection researchers because of the lack of techniques with which to access these populations A new molecular community profiling technique 16S rRNA genebased PCR followed by denaturing gradient gel electrophoresis (DGGE) has been used to analyse microbial communities in a number of environments and has many potential uses in plant protection research The technique is currently being used for the analysis of insect gut microflora characterisation of phylloplane and rhizosphere microbial populations and in environmental assessment of the effects of biopesticides and new technologies on indigenous soil microbes

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

The Synergy between CRISPR and Chemical Engineering

2019 ◽  
Vol 19 (3) ◽  
pp. 147-171
Author(s):  
Cia-Hin Lau ◽  
Chung Tin
Keyword(s):  
Viral Vectors ◽  
Chemical Engineering ◽  
Target Genes ◽  
New Technologies ◽  
Rapid Development ◽  
Genetic Circuits ◽  
Viral Packaging ◽  
Conditional Control ◽  
Logic Operations

Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.

scholarly journals Sequence Comparison of Vaginolysin from Different Gardnerella Species

Pathogens ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 86
Author(s):  
Erin M. Garcia ◽  
Myrna G. Serrano ◽  
Laahirie Edupuganti ◽  
David J. Edwards ◽  
Gregory A. Buck ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Microbiome ◽  
Human Microbiome Project ◽  
Distinct Species ◽  
Vaginal Swab ◽  
Metagenomic Data ◽  
Gardnerella Vaginalis ◽  
Vaginal Epithelial Cells ◽  
Species Specific

Gardnerella vaginalis has recently been split into 13 distinct species. In this study, we tested the hypotheses that species-specific variations in the vaginolysin (VLY) amino acid sequence could influence the interaction between the toxin and vaginal epithelial cells and that VLY variation may be one factor that distinguishes less virulent or commensal strains from more virulent strains. This was assessed by bioinformatic analyses of publicly available Gardnerella spp. sequences and quantification of cytotoxicity and cytokine production from purified, recombinantly produced versions of VLY. After identifying conserved differences that could distinguish distinct VLY types, we analyzed metagenomic data from a cohort of female subjects from the Vaginal Human Microbiome Project to investigate whether these different VLY types exhibited any significant associations with symptoms or Gardnerella spp.-relative abundance in vaginal swab samples. While Type 1 VLY was most prevalent among the subjects and may be associated with increased reports of symptoms, subjects with Type 2 VLY dominant profiles exhibited increased relative Gardnerella spp. abundance. Our findings suggest that amino acid differences alter the interaction of VLY with vaginal keratinocytes, which may potentiate differences in bacterial vaginosis (BV) immunopathology in vivo.

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