RNA Interference in Insects: From a Natural Mechanism of Gene Expression Regulation to a Biotechnological Crop Protection Promise.

Insect pests rank among the major limiting factors in agricultural production worldwide. In addition to direct effect on crops, some phytophagous insects are efficient vectors for plant disease transmission. Large amounts of conventional insecticides are required to secure food production worldwide, with a high impact on the economy and environment, particularly when beneficial insects are also affected by chemicals that frequently lack the desired specificity. RNA interference (RNAi) is a natural mechanism gene expression regulation and protection against exogenous and endogenous genetic elements present in most eukaryotes, including insects. Molecules of double-stranded RNA (dsRNA) or highly structured RNA are the substrates of cellular enzymes to produce several types of small RNAs (sRNAs), which play a crucial role in targeting sequences for transcriptional or post-transcriptional gene silencing. The relatively simple rules that underlie RNAi regulation, mainly based in Watson-Crick complementarity, have facilitated biotechnological applications based on these cellular mechanisms. This includes the promise of using engineered dsRNA molecules, either endogenously produced in crop plants or exogenously synthesized and applied onto crops, as a new generation of highly specific, sustainable, and environmentally friendly insecticides. Fueled on this expectation, this article reviews current knowledge about the RNAi pathways in insects, and some other applied questions such as production and delivery of recombinant RNA, which are critical to establish RNAi as a reliable technology for insect control in crop plants.

RNAi-mediated silencing of Mediterranean fruit fly (Ceratitis capitata) endogenous genes using orally-supplied double-stranded RNAs produced in Escherichia coli.

BACKGROUND: The Mediterranean fruit fly (medfly), Ceratitis capitata Wiedemann, is a major pest affecting fruit and vegetable production worldwide, whose control is mainly based on insecticides. Double-stranded RNA (dsRNA) able to down-regulate endogenous genes, thus affecting essential vital functions via RNA interference (RNAi) in pests and pathogens, is envisioned as a more specific and environmentally-friendly alternative to traditional insecticides. However, this strategy has not been explored in medfly yet. RESULTS: Here, we screened seven candidate target genes by injecting in adult medflies gene-specific dsRNA hairpins transcribed in vitro. Several genes were significantly down-regulated, resulting in increased insect mortality compared to flies treated with a control dsRNA targeting the green fluorescent protein (GFP) complementary DNA (cDNA). Three of the dsRNAs, homologous to the beta subunit of adenosine triphosphate (ATP) synthase (ATPsynbeta), a vacuolar ATPase (V-ATPase), and the ribosomal protein S13 (RPS13), were able to halve the probability of survival in only 48 h after injection. We then produced new versions of these three dsRNAs and that of the GFP control as circular molecules in Escherichia coli using a two-self-splicing-intron-based expression system and tested them as orally-delivered insecticidal compounds against medfly adults. We observed a significant down-regulation of V-ATPase and RPS13 messenger RNAs (mRNAs) (approximately 30% and 90%, respectively) compared with the control medflies after 3 days of treatment. No significant mortality was recorded in medflies, but egg laying and hatching reduction was achieved by silencing V-ATPase and RPS13. CONCLUSION: In sum, we report the potential of dsRNA molecules as oral insecticide in medfly. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Viroids: Non-Coding Circular RNAs Able to Autonomously Replicate and Infect Higher Plants.

Viroids are a unique type of infectious agent, exclusively composed of a relatively small (246-430 nt), highly base-paired, circular, non-coding RNA. Despite the small size and non-coding nature, the more-than-thirty currently known viroid species infectious of higher plants are able to autonomously replicate and move systemically through the host, thereby inducing disease in some plants. After recalling viroid discovery back in the late 60s and early 70s of last century and discussing current hypotheses about their evolutionary origin, this article reviews our current knowledge about these peculiar infectious agents. We describe the highly base-paired viroid molecules that fold in rod-like or branched structures and viroid taxonomic classification in two families, Pospiviroidae and Avsunviroidae, likely gathering nuclear and chloroplastic viroids, respectively. We review current knowledge about viroid replication through RNA-to-RNA rolling-circle mechanisms in which host factors, notably RNA transporters, RNA polymerases, RNases, and RNA ligases, are involved. Systemic movement through the infected plant, plant-to-plant transmission and host range are also discussed. Finally, we focus on the mechanisms of viroid pathogenesis, in which RNA silencing has acquired remarkable importance, and also for the initiation of potential biotechnological applications of viroid molecules.

Production of Recombinant RNA in Escherichia coli Using Eggplant Latent Viroid as a Scaffold.

Similar to viruses, viroids can also be engineered and transformed into useful biotechnological tools. We describe here a viroid-based system to produce large amounts of recombinant RNA in Escherichia coli. A precursor of eggplant latent viroid (ELVd), with the RNA of interest inserted between positions U245 and U246, is co-expressed in E. coli along the chloroplastic isoform of the eggplant tRNA ligase, the enzyme that mediates the circularization of this viroid in the infected plants. In the bacterial cells, the chimeric ELVd-RNA-of-interest precursor self-cleaves through the embedded hammerhead ribozymes, and the monomer is recognized and circularized by the co-expressed tRNA ligase. The resulting circular RNA, likely bound to the tRNA ligase, accumulates to a high concentration in the bacterial cells.

Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli.

RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 (DvSSJ1) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

Mutational Analysis of Eggplant Latent Viroid RNA Circularization by the Eggplant tRNA Ligase in Escherichia coli.

Eggplant latent viroid (ELVd) is a relatively small non-coding circular RNA that induces asymptomatic infections in eggplants (Solanum melongena L.). Like other viroid species that belong to the family Avsunviroidae, ELVd contains hammerhead ribozymes in the strands of both polarities that self-cleave RNAs producing terminal 5'-hydroxyl and 2',3'-cyclic phosphodiester groups. Available experimental data indicate that ELVd replicates in the chloroplasts of infected cells through a symmetric rolling-circle mechanism, in which RNA circularization is catalyzed by the chloroplastic isoform of the tRNA ligase. In this work, a mutational analysis was performed to gain insight into the sequence and structural requirements of the tRNA ligase-mediated circularization of ELVd RNAs. In the predicted minimum free energy conformation of the monomeric linear ELVd RNA intermediate of plus (+) polarity, the ligation site is located in the lower part of an opened internal loop, which is present in a quasi-rod-like structure that occupies the center of the molecule. The mutations analyzed herein consisted of punctual nucleotide substitutions and deletions surrounding the ligation site on the upper and lower strands of the ELVd quasi-double-stranded structure. Computational predictions of the mutated ELVd conformations indicated different degrees of distortions compared to the minimum free energy conformation of the wild-type ELVd linear monomer of + polarity. When these mutant RNAs were expressed in Escherichia coli, they were all circularized by the eggplant tRNA ligase with approximately the same efficiency as the wild-type ELVd, except for those that directly affected the ribozyme domain. These results suggest that the viroid ribozyme domains, in addition to self-cleavage, are also involved in the tRNA ligase-mediated circularization of the monomeric linear replication intermediates.