Reverse Genetics Tool Research Articles

Zebrafish research in Greece: swimming against the current.

The zebrafish is a vertebrate model extensively used in Developmental Biology and Human Disease modeling, as it shares high genetic and physiological similarities with humans. It has become the second most popular animal model, after mice, with several advantages over the latter: zebrafish are easily housed and cared for; the cost of installing and maintaining a zebrafish facility is significantly lower than for mice; and they reproduce often and develop quickly. Using zebrafish complies with the 3Rs principles of laboratory animal use. Zebrafish embryos develop externally and are transparent, allowing for in vivo non-invasive imaging. There are many transgenic and mutant lines available that mimic most human diseases, including reporter lines for most signaling pathways. There are also several reverse genetic tools to functionally verify genes or variants of unknown significance, identified in Genome-Wide Association Studies (GWAS) or using Next Generation Sequencing (NGS) approaches. In addition, the model emerges as an invaluable whole animal platform for various stages of drug discovery efforts by exploring the possibility of creating high-throughput phenotypic-driven screens. These include phenotypic screenings, determinations of general and/or specific toxicity (cardiac, renal, hepatotoxicity etc.), and mechanism of action studies. Finally, zebrafish are able to retain their capacity to regenerate most organs during their entire life span, making them a well-established model for the study of organ regeneration. The European Zebrafish Society consists of more than 180 research labs throughout Europe. In Greece however, zebrafish use remains rather limited. Here I present here a brief historical overview of zebrafish research in Greece.

RNAi Expression in Cotton for Control of Herbivorous Insects.

Cultivated cotton (Gossypium hirsutum) is heavily attacked by various species of insects worldwide and breeding of new varieties resistant to pests is still a hard battle to win. RNAi technology is an important reverse genetics tool to induce gene silencing in eukaryotic organisms and produce phenotypic modifications. In cotton, RNAi was applied to investigate gene function and enhance resistance to insects and pathogens. Different methods and techniques can be used to synthetize double stranded RNA (dsRNA) into plant cells. The Agrobacterium-mediated transformation is a common method to introduce RNAi binary plasmids into cotton genome and obtain stable transgenics plants. This methodology includes the coculture of cotton tissues with Agrobacterium cultures, selection of transgenic cells and induction of somatic embryogenesis to finally obtain transgenic plants after a relatively long period of time. The transient synthesis of dsRNA mediated by virus-induced gene silencing (VIGS) in cotton is an alternative to anticipate the silencing effect of a specific RNA sequence, prior to the development of a stable transgenic plant. VIGS vectors are incorporated into the plant by agroinfiltration technique. During VIGS replication inside plant cells, synthetized dsRNA allows the study on specific heterologous gene expression including the phenotypic effect on herbivorous target pests, thus facilitating a rapid evaluation of dsRNA expressed in cotton plants against individual insect target genes. Here we describe the complementation of these two techniques to evaluate RNAi-based cotton plant protection against insect pests.

Applications of RNA Interference in American Cockroach.

Cockroaches, a sanitary pest, are essential species in insect developmental and metamorphic studies due to their easy feeding and hemimetabolous characteristics. Altogether with well-annotated genome sequences, these advantages have made American cockroach, Periplaneta americana, an important hemimetabolous insect model. Limited by the shortage of knockout strategy, effective RNA interference (RNAi)-based gene knockdown becomes an indispensable technique in functional gene research of P. americana. The present protocol describes the RNAi operation techniques in P. americana. The protocol includes (1) selection of the P. americana at proper developmental stages, (2) preparation for the injection setting, (3) dsRNA injection, and (4) gene knockdown efficiency detection. RNAi is a powerful reverse genetic tool in P. americana. The majority of P. americana tissues are sensitive to extracellular dsRNA. Its simplicity allows researchers to quickly obtain dysfunctional phenotypes under one or multiple targeting dsRNA injections, enabling researchers to better use the P. americana for developmental and metamorphic studies.

Generation of duck Tembusu virus using a simple reverse genetic system in duck embryo fibroblast cells

Outbreaks of duck Tembusu virus (DTMUV) have caused serious economic losses in China since 2010. In this study, an infectious clone of the DTMUV BZ-2010strain, isolated from layer cherry duck in China, was constructed using the bacterium-free infectious subgenomic-amplicons method. The subgenomic-amplicons of the human cytomegalovirus promoter (pCMV) at the 5′ terminus of the first DNA fragment, the entire genome of DTMUV, and the hepatitis delta ribozyme followed by the simian virus 40 polyadenylation signal (HDR/SV40pA) at the 3′ terminus of the last DNA fragment were synthesized and amplified by PCR in three DNA fragments. The pCMV and HDR/SV40pA were used to drive the viral RNA transcription and generate a full-length RNA transcript of the virus, and were found to be effective in reassembling DTMUV in duck embryo fibroblast cells. The RNA transcripts from the infection clone were infectious in duck embryo fibroblast cells, generating the reconstituted DTMUV. This study provided a valuable reverse genetic tool for the further study DTMUV pathogenesis.

A cassava common mosaic virus vector for virus-induced gene silencing in cassava

BackgroundCassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing.ResultsIn this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months.ConclusionsThis CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

Targeted CRISPR-Cas9-based gene knockouts in the model brown alga Ectocarpus.

Brown algae are an important group of multicellular eukaryotes, phylogenetically distinct from both the animal and land plant lineages. Ectocarpus has emerged as a model organism to study diverse aspects of brown algal biology, but this system currently lacks an effective reverse genetics methodology to analyse the functions of selected target genes. Here, we report that mutations at specific target sites are generated following the introduction of CRISPR-Cas9 ribonucleoproteins into Ectocarpus cells, using either biolistics or microinjection as the delivery method. Individuals with mutations affecting the ADENINE PHOSPHORIBOSYL TRANSFERASE (APT) gene were isolated following treatment with 2-fluoroadenine, and this selection system was used to isolate individuals in which mutations had been introduced simultaneously at APT and at a second gene. This double mutation approach could potentially be used to isolate mutants affecting any Ectocarpus gene, providing an effective reverse genetics tool for this model organism. The availability of this tool will significantly enhance the utility of Ectocarpus as a model organism for this ecologically and economically important group of marine organisms. Moreover, the methodology described here should be readily transferable to other brown algal species.

A Methodological Advance of Tobacco Rattle Virus-Induced Gene Silencing for Functional Genomics in Plants.

As a promising high-throughput reverse genetic tool in plants, virus-induced gene silencing (VIGS) has already begun to fulfill some of this promise in diverse aspects. However, review of the technological advancements about widely used VIGS system, tobacco rattle virus (TRV)-mediated gene silencing, needs timely updates. Hence, this article mainly reviews viral vector construction, inoculation method advances, important influential factors, and summarizes the recent applications in diverse plant species, thus providing a better understanding and advice for functional gene analysis related to crop improvements.

PRAT Proteins Operate in Organellar Protein Import and Export in Arabidopsis thaliana.

Chloroplasts need to import preproteins and amino acids from the cytosol during their light-induced differentiation. Similarly, chloroplasts have to export organic matter including proteins and amino acids during leaf senescence. Members of the PRAT (preprotein and amino acid transporter) family are candidate transporters for both processes. Here, we defined the role of two small PRAT gene families, At4g26670 and At5g55510 (HP20 subfamily) versus At3g49560 and At5g24650 (HP30 subfamily) during greening of etiolated plants and during leaf senescence. Using a combination of reverse genetics, protein biochemistry and physiological tools, evidence was obtained for a role of chloroplast HP20, HP30 and HP30-2 in protein, but not amino acid, import into chloroplasts. HP20, HP30 and HP30-2 form larger complexes involved in the uptake of transit sequence-less cytosolic precursors. In addition, we identified a fraction of HP30-2 in mitochondria where it served a similar function as found for chloroplasts and operated in the uptake of transit sequence-less cytosolic precursor proteins. By contrast, HP22 was found to act in the export of proteins from chloroplasts during leaf senescence, and thus its role is entirely different from that of its orthologue, HP20. HP22 is part of a unique protein complex in the envelope of senescing chloroplasts that comprises at least 11 proteins and contains with HP65b (At5g55220) a protein that is related to the bacterial trigger factor chaperone. An ortholog of HP65b exists in the cyanobacterium Synechocystis and has previously been implicated in protein secretion. Whereas plants depleted of either HP22 or HP65b or even both were increasingly delayed in leaf senescence and retained much longer stromal chloroplast constituents than wild-type plants, HP22 overexpressors showed premature leaf senescence that was associated with accelerated losses of stromal chloroplast proteins. Together, our results identify the PRAT protein family as a unique system for importing and exporting proteins from chloroplasts.

Modeling Virus-Induced Inflammation in Zebrafish: A Balance Between Infection Control and Excessive Inflammation.

The inflammatory response to viral infection in humans is a dynamic process with complex cell interactions that are governed by the immune system and influenced by both host and viral factors. Due to this complexity, the relative contributions of the virus and host factors are best studied in vivo using animal models. In this review, we describe how the zebrafish (Danio rerio) has been used as a powerful model to study host-virus interactions and inflammation by combining robust forward and reverse genetic tools with in vivo imaging of transparent embryos and larvae. The innate immune system has an essential role in the initial inflammatory response to viral infection. Focused studies of the innate immune response to viral infection are possible using the zebrafish model as there is a 4-6 week timeframe during development where they have a functional innate immune system dominated by neutrophils and macrophages. During this timeframe, zebrafish lack a functional adaptive immune system, so it is possible to study the innate immune response in isolation. Sequencing of the zebrafish genome has revealed significant genetic conservation with the human genome, and multiple studies have revealed both functional conservation of genes, including those critical to host cell infection and host cell inflammatory response. In addition to studying several fish viruses, zebrafish infection models have been developed for several human viruses, including influenza A, noroviruses, chikungunya, Zika, dengue, herpes simplex virus type 1, Sindbis, and hepatitis C virus. The development of these diverse viral infection models, coupled with the inherent strengths of the zebrafish model, particularly as it relates to our understanding of macrophage and neutrophil biology, offers opportunities for far more intensive studies aimed at understanding conserved host responses to viral infection. In this context, we review aspects relating to the evolution of innate immunity, including the evolution of viral pattern recognition receptors, interferons and interferon receptors, and non-coding RNAs.

Quo Vadis Caenorhabditis elegans Metabolomics-A Review of Current Methods and Applications to Explore Metabolism in the Nematode.

Metabolomics and lipidomics recently gained interest in the model organism Caenorhabditis elegans (C. elegans). The fast development, easy cultivation and existing forward and reverse genetic tools make the small nematode an ideal organism for metabolic investigations in development, aging, different disease models, infection, or toxicology research. The conducted type of analysis is strongly depending on the biological question and requires different analytical approaches. Metabolomic analyses in C. elegans have been performed using nuclear magnetic resonance (NMR) spectroscopy, direct infusion mass spectrometry (DI-MS), gas-chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) or combinations of them. In this review we provide general information on the employed techniques and their advantages and disadvantages in regard to C. elegans metabolomics. Additionally, we reviewed different fields of application, e.g., longevity, starvation, aging, development or metabolism of secondary metabolites such as ascarosides or maradolipids. We also summarised applied bioinformatic tools that recently have been used for the evaluation of metabolomics or lipidomics data from C. elegans. Lastly, we curated metabolites and lipids from the reviewed literature, enabling a prototypic collection which serves as basis for a future C. elegans specific metabolome database.

Efficient flavinylation of glycosomal fumarate reductase by its own ApbE domain in Trypanosoma brucei.

A subset of flavoproteins has a covalently attached flavin prosthetic group enzymatically attached via phosphoester bonding. In prokaryotes, this is catalysed by alternative pyrimidine biosynthesis E (ApbE) flavin transferases. ApbE-like domains are present in few eukaryotic taxa, for example the N-terminal domain of fumarate reductase (FRD) of Trypanosoma, a parasitic protist known as a tropical pathogen causing African sleeping sickness. We use the versatile reverse genetic tools available for Trypanosoma to investigate the flavinylation of glycosomal FRD (FRDg) invivo in the physiological and organellar context. Using direct in-gel fluorescence detection of covalently attached flavin as proxy for activity, we show that the ApbE-like domain of FRDg has flavin transferase activity invivo. The ApbE domain is preceded by a consensus flavinylation target motif at the extreme N terminus of FRDg, and serine 9 in this motif is essential as flavin acceptor. The preferred mode of flavinylation in the glycosome was addressed by stoichiometric expression and comparison of native and catalytically inactive ApbE domains. In addition to the trans-flavinylation activity, the ApbE domain catalyses the intramolecular cis-flavinylation with at least fivefold higher efficiency. We discuss how the higher efficiency due to unusual fusion of the ApbE domain to its substrate protein FRD may provide a selective advantage by faster FRD biogenesis during rapid metabolic adaptation of trypanosomes. The first 37 amino acids of FRDg, including the consensus motif, are sufficient as flavinylation target upon fusion to other proteins. We propose FRDg(1-37) as 4-kDa heat-stable, detergent-resistant fluorescent protein tag and suggest its use as a new tool to study glycosomal protein import.

Development of Rice Stripe Tenuivirus Minireplicon Reverse Genetics Systems Suitable for Analyses of Viral Replication and Intercellular Movement.

Rice stripe virus (RSV), a tenuivirus with four negative-sense/ambisense genome segments, is one of the most devastating viral pathogens affecting rice production in many Asian countries. Despite extensive research, our understanding of RSV infection cycles and pathogenesis has been severely impaired by the lack of reverse genetics tools. In this study, we have engineered RSV minireplicon (MR)/minigenome cassettes with reporter genes substituted for the viral open reading frames in the negative-sense RNA1 or the ambisense RNA2-4 segments. After delivery to Nicotiana benthamiana leaves via agroinfiltration, MR reporter gene expression was detected only when the codon-optimized large viral RNA polymerase protein (L) was coexpressed with the nucleocapsid (N) protein. MR activity was also critically dependent on the coexpressed viral suppressors of RNA silencing, but ectopic expression of the RSV-encoded NS3 silencing suppressor drastically decreased reporter gene expression. We also developed intercellular movement-competent MR systems with the movement protein expressed either in cis from an RNA4-based MR or in trans from a binary plasmid. Finally, we generated multicomponent replicon systems by expressing the N and L proteins directly from complementary-sense RNA1 and RNA3 derivatives, which enhanced reporter gene expression, permitted autonomous replication and intercellular movement, and reduced the number of plasmids required for delivery. In summary, this work enables reverse genetics analyses of RSV replication, transcription, and cell-to-cell movement and provides a platform for engineering more complex recombinant systems.

Efficient deletion of multiple circle RNA loci by CRISPR-Cas9 reveals Os06circ02797 as a putative sponge for OsMIR408 in rice.

SummaryCRISPR‐Cas9 is an emerging genome editing tool for reverse genetics in plants. However, its application for functional study of non‐coding RNAs in plants is still at its infancy. Despite being a major class of non‐coding RNAs, the biological roles of circle RNAs (circRNAs) remain largely unknown in plants. Previous plant circRNA studies have focused on identification and annotation of putative circRNAs, with their functions largely uninvestigated by genetic approaches. Here, we applied a multiplexed CRISPR‐Cas9 strategy to efficiently acquire individual null mutants for four circRNAs in rice. We showed each of these rice circRNA loci (Os02circ25329, Os06circ02797, Os03circ00204 and Os05circ02465) can be deleted at 10% or higher efficiency in both protoplasts and stable transgenic T0 lines. Such high efficiency deletion enabled the generation of circRNA null allele plants without the CRISPR‐Cas9 transgene in the T1 generation. Characterization of the mutants reveals these circRNAs’ participation in salt stress response during seed germination and in particular the Os05circ02465 null mutant showed high salt tolerance. Notably, the seedlings of the Os06circ02797 mutant showed rapid growth phenotype after seed germination with the seedlings containing higher chlorophyll A/B content. Further molecular and computational analyses suggested a circRNA–miRNA–mRNA regulatory network where Os06circ02797 functions to bind and sequester OsMIR408, an important and conserved microRNA in plants. This study not only presents genetic evidence for the first time in plants that certain circRNAs may serve as sponges to negatively regulate miRNAs, a phenomenon previously demonstrated in mammalian cells, but also provides important insights for improving agronomic traits through gene editing of circRNA loci in crops.

Development of EMS-induced Mutagenized Groundnut Population and Discovery of Point Mutations in the <i>ahFAD2</i> and <i>Ara h 1</i> Genes by TILLING

Reducing allergenicity and increasing oleic content are important goals in groundnut breeding studies. Ara h 1 is a major allergen gene and Delta(12)-fatty-acid desaturase (FAD2) is responsible for converting oleic into linoleic acid. These genes have homoeologues with one copy in each subgenome, identified as Ara h 1.01, Ara h 1.02, ahFAD2A and ahFAD2B in tetraploid groundnut. To alter functional properties of these genes we have generated an Ethyl Methane Sulfonate (EMS) induced mutant population to be used in Targeting Induced Local Lesions in Genomes (TILLING) approach. Seeds were exposed to two EMS concentrations and the germination rates were calculated as 90.1% (1353 plants) for 0.4% and 60.4% (906 plants) for 1.2% EMS concentrations in the M1 generation. Among the 1541 M2 mutants, 768 were analyzed by TILLING using four homoeologous genes. Two heterozygous mutations were identified in the ahFAD2B and ahFAD2A gene regions from 1.2% and 0.4% EMS-treated populations, respectively. The mutation in ahFAD2B resulted in an amino acid change, which was serine to threonine predicted to be tolerated according to SIFT analysis. The other mutation causing amino acid change, glycine to aspartic acid was predicted to affect protein function in ahFAD2A. No mutations were detected in Ara h 1.01 and Ara h 1.02 for both EMS-treatments after sequencing. We estimated the overall mutation rate to be 1 mutation every 2139 kb. The mutation frequencies were also 1/317 kb for ahFAD2A in 0.4% EMS and 1/466 kb for ahFAD2B in 1.2% EMS treatments. The results demonstrated that TILLING is a powerful tool to interfere with gene function in crops and the mutagenized population developed in this study can be used as an efficient reverse genetics tool for groundnut improvement and functional genomics.

Short term transcriptional responses of P450s to phytochemicals in insects and mites.

Cytochrome P450 monooxygenases (P450s) play a key role in the detoxification of phytochemicals in arthropod herbivores. We present here an overview of recent progress in understanding the breadth and specificity of gene expression plasticity of P450s in response to phytochemicals. We discuss experimental setups and new findings in mechanisms of P450 regulation. Whole genome transcriptomic analysis of arthropod herbivores, either after direct administration of phytochemicals or after host plant shifts, allowed to integrate various levels of chemical complexity and lead to the unbiased identification of responsive P450 genes. However, despite progress in identification of inducible P450s, the link between induction and metabolism is still largely unexplored, and to what extent the overall response is biologically functional should be further investigated. In the near future, such studies will be more straightforward as forward and reverse genetic tools become more readily available.

RNA Interference (RNAi) in Tomato Crop Research.

RNA interference (RNAi) is a posttranscriptional gene silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in many organisms. In tomato, RNAi technology has widely been used as a reverse genetic tool for functional genomics study. Generally, RNAi is often achieved through transgenes producing hairpin RNA molecules. RNAi lines have the advantage with respect to more modern CRISPR/Cas9 mutants of different levels of downregulation of target gene, and allow the characterization of life-essential genes that cannot be knocked out without killing the organism. Also, RNAi allows to suppress gene expression in multigene families in a regulated manner. In this chapter, an efficient approach to create RNAi stable knockdown-transformed tomato lines is reported. In order, it describes the choice of the target silencing fragment, a highly efficient cloning strategy for the hairpin RNA construct production, a relatively easy procedure to transform and regenerate tomato plants using Agrobacterium tumefaciens and a methodology to test the goodness of the transformation procedure.

A functional genomics approach to dissect spotted alfalfa aphid resistance in Medicago truncatula

Aphids are virus-spreading insect pests affecting crops worldwide and their fast population build-up and insecticide resistance make them problematic to control. Here, we aim to understand the molecular basis of spotted alfalfa aphid (SAA) or Therioaphis trifolii f. maculata resistance in Medicago truncatula, a model organism for legume species. We compared susceptible and resistant near isogenic Medicago lines upon SAA feeding via transcriptome sequencing. Expression of genes involved in defense and stress responses, protein kinase activity and DNA binding were enriched in the resistant line. Potentially underlying some of these changes in gene expression was the finding that members of the MYB, NAC, AP2 domain and ERF transcription factor gene families were differentially expressed in the resistant versus susceptible lines. A TILLING population created in the resistant cultivar was screened using exome capture sequencing and served as a reverse genetics tool to functionally characterise genes involved in the aphid resistance response. This screening revealed three transcription factors (a NAC, AP2 domain and ERF) as important regulators in the defence response, as a premature stop-codon in the resistant background led to a delay in aphid mortality and enhanced plant susceptibility. This combined functional genomics approach will facilitate the future development of pest resistant crops by uncovering candidate target genes that can convey enhanced aphid resistance.

RNAi-Mediated Suppression of Laccase2 Impairs Cuticle Tanning and Molting in the Cotton Boll Weevil (Anthonomus grandis).

The cotton boll weevil, Anthonomus grandis, is the most economically important pest of cotton in Brazil. Pest management programs focused on A. grandis are based mostly on the use of chemical insecticides, which may cause serious ecological impacts. Furthermore, A. grandis has developed resistance to some insecticides after their long-term use. Therefore, alternative control approaches that are more sustainable and have reduced environmental impacts are highly desirable to protect cotton crops from this destructive pest. RNA interference (RNAi) is a valuable reverse genetics tool for the investigation of gene function and has been explored for the development of strategies to control agricultural insect pests. This study aimed to evaluate the biological role of the Laccase2 (AgraLac2) gene in A. grandis and its potential as an RNAi target for the control of this insect pest. We found that AgraLac2 is expressed throughout the development of A. grandis with significantly higher expression in pupal and adult developmental stages. In addition, the immunolocalization of the AgraLac2 protein in third-instar larvae using specific antibodies revealed that AgraLac2 is distributed throughout the epithelial tissue, the cuticle and the tracheal system. We also verified that the knockdown of AgraLac2 in A. grandis resulted in an altered cuticle tanning process, molting defects and arrested development. Remarkably, insects injected with dsAgraLac2 exhibited defects in cuticle hardening and pigmentation. As a consequence, the development of dsAgraLac2-treated insects was compromised, and in cases of severe phenotypic defects, the insects subsequently died. On the contrary, insects subjected to control treatments did not show any visible phenotypic defects in cuticle formation and successfully molted to the pupal and adult stages. Taken together, our data indicate that AgraLac2 is involved in the cuticle tanning process in A. grandis and may be a promising target for the development of RNAi-based technologies.

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

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.

E-cadherin focuses protrusion formation at the front of migrating cells by impeding actin flow

The migration of many cell types relies on the formation of actomyosin-dependent protrusions called blebs, but the mechanisms responsible for focusing this kind of protrusive activity to the cell front are largely unknown. Here, we employ zebrafish primordial germ cells (PGCs) as a model to study the role of cell-cell adhesion in bleb-driven single-cell migration in vivo. Utilizing a range of genetic, reverse genetic and mathematical tools, we define a previously unknown role for E-cadherin in confining bleb-type protrusions to the leading edge of the cell. We show that E-cadherin-mediated frictional forces impede the backwards flow of actomyosin-rich structures that define the domain where protrusions are preferentially generated. In this way, E-cadherin confines the bleb-forming region to a restricted area at the cell front and reinforces the front-rear axis of migrating cells. Accordingly, when E-cadherin activity is reduced, the bleb-forming area expands, thus compromising the directional persistence of the cells.