Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso)4E in Beta vulgaris.

Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant-virus interaction. Most of these plant-virus interactions were found between plant eIFs and the viral protein genome-linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv-eIF(iso)4E as a susceptibility factor towards the VPg-carrying beet chlorosis virus (genus Polerovirus). Using yeast two-hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv-eIF(iso)4E and the putative BChV-VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv-eIF4E and Bv-eIF(iso)4E. These VPg-eIF interactions within the polerovirus-beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap-binding pocket of Bv-eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B. vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. A simultaneous knockout of the identified BMYV-interaction partners Bv-eIF4E and Bv-eIF(iso)4E was not achieved, but Bv-eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet.

The Study of Cell-Penetrating Peptides to Deliver dsRNA and siRNA by Feeding in the Desert Locust, Schistocerca gregaria.

RNA(i) interference is a gene silencing mechanism triggered by double-stranded (ds)RNA, which promises to contribute to species-specific insect pest control strategies. The first step toward the application of RNAi as an insecticide is to enable efficient gene silencing upon dsRNA oral delivery. The desert locust, Schistocerca gregaria is a devastating agricultural pest. While this species is responsive to dsRNA delivered by intra-hemocoelic injection, it is refractory to orally delivered dsRNA. In this study, we evaluated the capacity of five cell-penetrating peptides (CPPs) to bind long dsRNA and protect it from the locust midgut environment. We then selected the CPP EB1 for further in vivo studies. EB1:dsRNA complexes failed to induce RNAi by feeding. Interestingly, we observed that intra-hemocoelic injection of small-interfering (si)RNAs does not result in a silencing response, but that this response can be obtained by injecting EB1:siRNA complexes. EB1 also protected siRNAs from midgut degradation activity. However, EB1:siRNA complexes failed as well in triggering RNAi when fed. Our findings highlight the complexity of the dsRNA/siRNA-triggered RNAi in this species and emphasize the multifactorial nature of the RNAi response in insects. Our study also stresses the importance of in vivo studies when it comes to dsRNA/siRNA delivery systems.

Identification and profiling of stable microRNAs in hemolymph of young and old Locusta migratoria fifth instars.

Since the discovery of the first microRNA (miRNA) in the nematode Caenorhabditis elegans, numerous novel miRNAs have been identified which can regulate presumably every biological process in a wide range of metazoan species. In accordance, several insect miRNAs have been identified and functionally characterized. While regulatory RNA pathways are traditionally described at an intracellular level, studies reporting on the presence and potential role of extracellular (small) sRNAs have been emerging in the last decade, mainly in mammalian systems. Interestingly, evidence in several species indicates the functional transfer of extracellular RNAs between donor and recipient cells, illustrating RNA-based intercellular communication. In insects, however, reports on extracellular small RNAs are emerging but the number of detailed studies is still very limited. Here, we demonstrate the presence of stable sRNAs in the hemolymph of the migratory locust, Locusta migratoria. Moreover, the levels of several extracellular miRNAs (ex-miRNAs) present in locust hemolymph differed significantly between young and old fifth nymphal instars. In addition, we performed a 'proof of principle' experiment which suggested that extracellularly delivered miRNA molecules are capable of affecting the locusts' development.

PIWI Proteins Play an Antiviral Role in Lepidopteran Cell Lines.

Insect antiviral immunity primarily relies on RNAi mechanisms. While a key role of small interfering (si)RNAs and AGO proteins has been well established in this regard, the situation for PIWI proteins and PIWI-interacting (pi)RNAs is not as clear. In the present study, we investigate whether PIWI proteins and viral piRNAs are involved in the immunity against single-stranded RNA viruses in lepidopteran cells, where two PIWIs are identified (Siwi and Ago3). Via loss- and gain-of-function studies in Bombyx mori BmN4 cells and in Trichoplusia ni High Five cells, we demonstrated an antiviral role of Siwi and Ago3. However, small RNA analysis suggests that viral piRNAs can be absent in these lepidopteran cells. Together with the current literature, our results support a functional diversification of PIWI proteins in insects.

Accelerated delivery of dsRNA in lepidopteran midgut cells by a Galanthus nivalis lectin (GNA)-dsRNA-binding domain fusion protein.

Lepidopteran insects are highly refractory to oral RNA interference (RNAi). Degradation, impaired cellular uptake and intracellular transport of double-stranded RNA (dsRNA) are considered the major factors responsible for the reduced RNAi efficiency in these insects. In this study, the potential of lectins to improve dsRNA delivery and RNAi efficacy was evaluated. First, a fusion protein consisting of the Galanthus nivalis agglutinin (GNA) and a dsRNA binding domain was developed, further referred to as GNA:dsRBD (GNAF). Then, its ability to increase dsRNA uptake and transfection efficiency in lepidopteran midgut cells was evaluated, as well as its ability to protect and promote the RNAi response in the beet armyworm Spodoptera exigua. Confocal microscopy analysis showed that GNAF-complexed dsRNA was internalized faster in Choristoneura fumiferana midgut CF1 cells (1 min) compared to naked dsRNA (>1 h). The faster uptake was also correlated with an increased RNAi efficiency in these CF1 cells. In vivo feeding bioassays with GNAF-complexed dsRNA led to an increased mortality in S. exigua compared to the controls. By targeting the essential gene V-ATPase A, we observed that the mortality increased to 48% in the GNAF-dsRNA treatment compared to only 8.3% and 6.6% in the control treatments with the naked dsRNA and the GNAF, respectively.

Extracellular vesicles spread the RNA interference signal of Tribolium castaneum TcA cells.

The potential utility of RNA interference (RNAi) to control insect pests and viral infections depends largely on the target organism's ability to systemically spread the RNAi response. The efficacy of systemic RNAi varies among insects, though it has been shown to be high in the red flour beetle, Tribolium castaneum. We identified an extracellular RNAi signal that is present in the culture medium of T. castaneum (TcA) cells after treatment with long dsRNA specific for a luciferase reporter gene. Luciferase-specific siRNAs were detected in extracellular vesicles (EVs) that were purified from the culture medium of these dsRNA-treated cells. Furthermore, by measuring the silencing of luciferase expression, we showed that these siRNA-containing EVs can act as an RNAi signal for recipient TcA cells. We have therefore shown that a systemic RNAi response upon dsRNA treatment can be effectively spread through EVs.

Generation of Virus- and dsRNA-Derived siRNAs with Species-Dependent Length in Insects.

Double-stranded RNA (dsRNA) molecules of viral origin trigger a post-transcriptional gene-silencing mechanism called RNA interference (RNAi). Specifically, virally derived dsRNA is recognized and cleaved by the enzyme Dicer2 into short interfering RNAs (siRNAs), which further direct sequence-specific RNA silencing, ultimately silencing replication of the virus. Notably, RNAi can also be artificially triggered by the delivery of gene-specific dsRNA, thereby leading to endogenous gene silencing. This is a widely used technology that holds great potential to contribute to novel pest control strategies. In this regard, research efforts have been set to find methods to efficiently trigger RNAi in the field. In this article, we demonstrate the generation of dsRNA- and/or virus-derived siRNAs-the main RNAi effectors-in six insect species belonging to five economically important orders (Lepidoptera, Orthoptera, Hymenoptera, Coleoptera, and Diptera). In addition, we describe that the siRNA length distribution is species-dependent. Taken together, our results reveal interspecies variability in the (antiviral) RNAi mechanism in insects and show promise to contribute to future research on (viral-based) RNAi-triggering mechanisms in this class of animals.

PIWI pathway against viruses in insects.

Piwi-interacting RNAs (piRNAs) are an animal-specific class of small non-coding RNAs that are generated via a biogenesis pathway distinct from small interfering RNAs (siRNAs) and microRNAs (miRNAs). There are variations in piRNA biogenesis that depend on several factors, such as the cell type (germline or soma), the organism, and the purpose for which they are being produced, such as transposon-targeting, viral-targeting, or gene-derived piRNAs. Interestingly, the genes involved in the PIWI/piRNA pathway are more rapidly evolving compared with other RNA interference (RNAi) genes. In this review, the role of the piRNA pathway in the antiviral response is reviewed based on recent findings in insect models such as Drosophila, mosquitoes, midges and the silkworm, Bombyx mori. We extensively discuss the special features that characterize host-virus piRNA responses with respect to the proteins and the genes involved, the viral piRNAs' sequence characteristics, the target strand orientation biases as well as the viral piRNA target hotspots across the viral genomes. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > RNAi: Mechanisms of Action Regulatory RNAs/RNAi/Riboswitches > Biogenesis of Effector Small RNAs.

The presence of extracellular microRNAs in the media of cultured Drosophila cells.

While regulatory RNA pathways, such as RNAi, have commonly been described at an intracellular level, studies investigating extracellular RNA species in insects are lacking. In the present study, we demonstrate the presence of extracellular microRNAs (miRNAs) in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), we analysed the presence of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates, obtained from the cell-free conditioned media of S2 and Cl.8 cell cultures. Next-generation RNA-sequencing data confirmed our qRT-PCR results and provided evidence for selective miRNA secretion in EVs. To our knowledge, this is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects, the most speciose group of animals.

Insights into RNAi-based antiviral immunity in Lepidoptera: acute and persistent infections in Bombyx mori and Trichoplusia ni cell lines.

The control of viral infections in insects is a current issue of major concern and RNA interference (RNAi) is considered the main antiviral immune response in this group of animals. Here we demonstrate that overexpression of key RNAi factors can help to protect insect cells against viral infections. In particular, we show that overexpression of Dicer2 and Argonaute2 in lepidopteran cells leads to improved defense against the acute infection of the Cricket Paralysis Virus (CrPV). We also demonstrate an important role of RNAi in the control of persistent viral infections, as the one caused by the Macula-like Latent Virus (MLV). Specifically, a direct interaction between Argonaute2 and virus-specific small RNAs is shown. Yet, while knocking down Dicer2 and Argonaute2 resulted in higher transcript levels of the persistently infecting MLV in the lepidopteran cells under investigation, overexpression of these proteins could not further reduce these levels. Taken together, our data provide deep insight into the RNAi-based interactions between insects and their viruses. In addition, our results suggest the potential use of an RNAi gain-of-function approach as an alternative strategy to obtain reduced viral-induced mortality in Lepidoptera, an insect order that encompasses multiple species of relevant economic value.

The evolution of animal Argonautes: evidence for the absence of antiviral AGO Argonautes in vertebrates.

In addition to mediating regulation of endogenous gene expression, RNA interference (RNAi) in plants and invertebrates plays a crucial role in defense against viruses via virus-specific siRNAs. Different studies have demonstrated that the functional diversity of RNAi in animals is linked to the diversification of the Argonaute superfamily, central components of RISCs (RNA induced silencing complexes). The animal Argonaute superfamily is traditionally grouped into AGO and PIWI Argonautes. Yet, by performing phylogenetic analyses and determining the selective evolutionary pressure in the metazoan Argonaute superfamily, we provide evidence for the existence of three conserved Argonaute lineages between basal metazoans and protostomes, namely siRNA-class AGO, miRNA-class AGO and PIWI Argonautes. In addition, it shown that the siRNA-class AGO lineage is characterized by high rates of molecular evolution, suggesting a role in the arms race with viruses, while the miRNA-class AGOs display strong sequence conservation. Interestingly, we also demonstrate that vertebrates lack siRNA-class AGO proteins and that vertebrate AGOs display low rates of molecular evolution. In this way, we provide supportive evidence for the loss of the antiviral siRNA-class AGO group in vertebrates and discuss the consequence hereof on antiviral immunity and the use of RNAi as a loss of function tool in these animals.

Knockdown of nuclease activity in the gut enhances RNAi efficiency in the Colorado potato beetle, Leptinotarsa decemlineata, but not in the desert locust, Schistocerca gregaria.

The responsiveness towards orally delivered dsRNA and the potency of a subsequent environmental RNA interference (RNAi) response strongly differs between different insect species. While some species are very sensitive to dsRNA delivery through the diet, others are not. The underlying reasons for this may vary, but degradation of dsRNA by nucleases in the gut lumen is believed to play a crucial role. The Colorado potato beetle, Leptinotarsa decemlineata, is a voracious defoliator of potato crops worldwide, and is currently under investigation for novel control methods based on dsRNA treatments. Here we describe the identification and characterization of two nuclease genes exclusively expressed in the gut of this pest species. Removal of nuclease activity in adults increased the sensitivity towards dsRNA and resulted in improved protection of potato plants. A similar strategy in the desert locust, Schistocerca gregaria, for which we show a far more potent nuclease activity in the gut juice, did however not lead to an improvement of the RNAi response. Possible reasons for this are discussed. Taken together, the present data confirm a negative effect of nucleases in the gut on the environmental RNAi response, and further suggest that interfering with this activity is a strategy worth pursuing for improving RNAi efficacy in insect pest control applications.

Persistent RNA virus infection of lepidopteran cell lines: Interactions with the RNAi machinery.

RNAi is broadly used as a technique for specific gene silencing in insects but few studies have investigated the factors that can affect its efficiency. Viral infections have the potential to interfere with RNAi through their production of viral suppressors of RNAi (VSRs) and the production of viral small RNAs that can saturate and inactivate the RNAi machinery. In this study, the impact of persistent infection of the RNA viruses Flock house virus (FHV) and Macula-like virus (MLV) on RNAi efficiency was investigated in selected lepidopteran cell lines. Lepidopteran cell lines were found to be readily infected by both viruses without any apparent pathogenic effects, with the exception of Bombyx-derived Bm5 and BmN4 cells, which could not be infected by FHV. Because Sf21 cells were free from both FHV and MLV and Hi5-SF were free from FHV and only contained low levels of MLV, they were tested to evaluate the impact of the presence of the virus. Two types of RNAi reporter assays however did not detect a significant interference with gene silencing in infected Sf21 and Hi5-SF cells when compared to virus-free cells. In Hi5 cells, the presence of FHV could be easily cleared through the expression of an RNA hairpin that targets its VSR gene, confirming that the RNAi mechanism was not inhibited. Sequencing indicated that the B2 RNAi inhibitor gene of FHV and a putative VSR gene from MLV were intact in persistently infected cell lines, indicating that protection against RNAi remains essential for virus survival. It is proposed that infection levels of persistent viruses in the cell lines are too low to have an impact on RNAi efficiency in the lepidopteran cell lines and that encoded VSRs act locally at the sites of viral replication (mitochondrial membranes) without affecting the rest of the cytoplasm.

Drosha, Dicer-1 and Argonaute-1 in the desert locust: phylogenetic analyses, transcript profiling and regulation during phase transition and feeding.

In this article, we identify and characterise the miRNA machinery components Drosha, Dicer-1 and Argonaute-1 of the desert locust. By means of phylogenetic analyses, we reveal important insights in the evolutionary context of these components. Our data illustrate that insect Argonaute-1 proteins form a monophyletic group with ALG-1 and ALG-2 of Caenorhabditis elegans and with the four (non-Piwi) Argonaute proteins present in humans. On the other hand, humans apparently lack clear homologues of the insect Argonaute-2 proteins. In addition, we demonstrate that drosha, dicer-1 and argonaute-1 display wide transcript tissue-distribution in adult desert locusts, and that during locust phase transition and feeding of starved locusts the expression levels of the miRNA pathway are regulated at the transcript level.

Biological mechanisms determining the success of RNA interference in insects.

Insects constitute the largest group of animals on this planet, having a huge impact on our environment, as well as on our quality of life. RNA interference (RNAi) is a posttranscriptional gene silencing mechanism triggered by double-stranded (ds)RNA fragments. This process not only forms the basis of a widely used reverse genetics research method in many different eukaryotes but also holds great promise to contribute to the species-specific control of agricultural pests and to combat viral infections in beneficial and disease vectoring insects. However, in many economically important insect species, such as flies, mosquitoes, and caterpillars, systemic delivery of naked dsRNA does not trigger effective gene silencing. Although many components of the RNAi pathway have initially been deciphered in the fruit fly, Drosophila melanogaster, it will be of major importance to investigate this process in a wider variety of species, including dsRNA-sensitive insects such as locusts and beetles, to elucidate the factors responsible for the remarkable variability in RNAi efficiency, as observed in different insects. In this chapter, we review the current knowledge on the RNAi pathway, as well as the most recent insights into the mechanisms that might determine successful RNAi in insects.

Lipophorins can adhere to dsRNA, bacteria and fungi present in the hemolymph of the desert locust: a role as general scavenger for pathogens in the open body cavity.

Desert locusts are characterized by a highly sensitive and effective RNA interference (RNAi) response. Moreover, delivery of dsRNA into the open body cavity will elicit potent silencing effects throughout the body. On the other hand, many other insect species, such as Bombyx mori and Drosophila melanogaster, lack the ability to efficiently spread the RNAi-signal. In this study, we demonstrated that, in the serum of the desert locust, lipophorins adhere to dsRNA-fragments. Lipophorins can be subdivided into high density and low density lipophorins (HDLp and LDLp), according to their buoyant density, and we showed that both types of lipophorins can interact with dsRNA fragments. Furthermore, in the presence of (gram-positive) bacteria or fungi, LDLp induce the formation of pathogen aggregates, while no clear aggregation effects were detected in the presence of HDLp.

Effects of different dietary conditions on the expression of trypsin- and chymotrypsin-like protease genes in the digestive system of the migratory locust, Locusta migratoria.

While technological advancements have recently led to a steep increase in genomic and transcriptomic data, and large numbers of protease sequences are being discovered in diverse insect species, little information is available about the expression of digestive enzymes in Orthoptera. Here we describe the identification of Locusta migratoria serine protease transcripts (cDNAs) involved in digestion, which might serve as possible targets for pest control management. A total of 5 putative trypsin and 15 putative chymotrypsin gene sequences were characterized. Phylogenetic analysis revealed that these are distributed among 3 evolutionary conserved clusters. In addition, we have determined the relative gene expression levels of representative members in the gut under different feeding conditions. This study demonstrated that the transcript levels for all measured serine proteases were strongly reduced after starvation. On the other hand, larvae of L. migratoria displayed compensatory effects to the presence of Soybean Bowman Birk (SBBI) and Soybean Trypsin (SBTI) inhibitors in their diet by differential upregulation of multiple proteases. A rapid initial upregulation was observed for all tested serine protease transcripts, while only for members belonging to class I, the transcript levels remained elevated after prolonged exposure. In full agreement with these results, we also observed an increase in proteolytic activity in midgut secretions of locusts that were accustomed to the presence of protease inhibitors in their diet, while no change in sensitivity to these inhibitors was observed. Taken together, this paper is the first comprehensive study on dietary dependent transcript levels of proteolytic enzymes in Orthoptera. Our data suggest that compensatory response mechanisms to protease inhibitor ingestion may have appeared early in insect evolution.

Identification, functional characterization and phylogenetic analysis of double stranded RNA degrading enzymes present in the gut of the desert locust, Schistocerca gregaria.

RNA interference (RNAi) has become a widely used reverse genetics tool in eukaryotes and holds great potential to contribute to the development of novel strategies for insect pest control. While previous studies clearly demonstrated that injection of dsRNA into the body cavity of the desert locust, Schistocerca gregaria, is highly effective to induce gene silencing effects, we observed that the RNAi response is much less sensitive to orally delivered dsRNA. In line with this, we report on the presence of a potent dsRNA degrading activity in the midgut juice. Four different dsRNase sequences that belong to the DNA/RNA Non-specific Nuclease superfamily were retrieved from a transcriptome database of the desert locust. Surprisingly, we have found that, in the publicly available eukaryote nucleotide sequence databases, the presence of this group of enzymes is restricted to insects and crustaceans. Nonetheless, phylogenetic analyses predict a common origin of these enzymes with the Endonuclease G (EndoG) Non-specific Nucleases that display a widespread taxonomic distribution. Moreover, in contrast to the Sg-endoG transcript, the four Sg-dsRNase transcripts appear to be specifically expressed in the gut. Finally, by means of RNAi, we provide evidence for an important contribution of dsRNase2 to the dsRNA degrading activity that is present in the gut lumen of S. gregaria.

Systemic RNA interference in locusts: reverse genetics and possibilities for locust pest control.

RNA interference (RNAi) is a biological process triggered by double stranded (ds)RNA that results in sequence-dependent mRNA degradation. Because of its high specificity, this post-transcriptional gene silencing mechanism is a widely used tool for reverse genetics in several insect species. In particular, locusts possess a very robust and sensitive RNAi response that has already been exploited to investigate a diverse range of important physiological processes. These orthopteran insects constitute important model organisms in several areas of entomology, but they can also become voracious swarming pests that threaten the agricultural production in large parts of the world. In comparison to the widely applied chemical insecticides, the RNAi-technology could contribute to the development of a novel generation of insecticides, with high species-specificity. In this article, we discuss the potential of the RNAi-technology in loss of function studies in locusts, as well as to control locust populations.

In vivo effect of Neuropeptide F on ecdysteroidogenesis in adult female desert locusts (Schistocerca gregaria).

Neuropeptides are important regulatory factors that mediate key life processes, both in vertebrates and invertebrates. Many insect neuropeptides display pleiotropic activities, which means that they can influence multiple aspects of insect physiology. In the fruit fly, Drosophila melanogaster, Neuropeptide F (NPF) mediates diverse physiological processes, such as learning, stress responses, feeding and male courtship behavior. In locusts, only a truncated form of the predicted "full-length" NPF, the nonapeptide "trNPF", has been isolated. This nonapeptide previously proved to be biologically active, since it was shown to influence food intake and weight increase, as well as oocyte growth in adult female desert locusts (Schistocerca gregaria [Forskål]). In the present study, we have further analyzed the effect of trNPF on female reproductive physiology in S. gregaria. We confirmed that daily trNPF injections in adult females elicit an increase of oocyte size. In addition, an RNAi-mediated knockdown of the Schgr-NPF precursor transcript in adult female locusts resulted in the opposite effect, i.e. significantly smaller oocytes. Moreover, we discovered that daily injections of trNPF in adult female S. gregaria, caused higher ecdysteroid titers in the ovaries and accelerated the appearance of ecdysteroid peaks in the hemolymph of these animals. The RNAi-based knockdown of the Schgr-NPF precursor transcript clearly resulted in reduction of both hemolymph and ovarian ecdysteroid concentrations, confirming the stimulatory effects of trNPF injections on adult female ecdysteroid levels. The observed results are discussed in relation to previous reports on NPF activities in locusts and other insects.