Methods for Delivery of dsRNAs for Agricultural Pest Control: The Case of Lepidopteran Pests.

RNA interference (RNAi) is a natural mechanism of gene regulation, highly conserved in eukaryotes. Since the elucidation of the gene silencing mechanism, RNAi became an important tool used in insect reverse genetics. The demonstration of effective target-gene silencing by ingestion of double-stranded RNA (dsRNA) produced by transgenic plants indicated the RNAi potential to be used in insect pest management, particularly in agriculture. However, the efficiency of gene silencing by RNAi in insects may vary according to the target taxa, and lepidopteran species have been shown to be quite recalcitrant to RNAi. Developing transgenic plants is a time-consuming and labor-intensive process, so alternative oral delivery systems are required to develop and optimize RNAi settings, such as selecting an efficient target gene, and dsRNA design, length, and stability, among other features. We have developed delivery systems to evaluate dsRNAs to silence genes from two important lepidopteran crop pests of tomato (Solanum lycopersicum) and sugarcane (Saccharum × officinarum): Tuta absoluta (Meyrick), the South American Tomato Pinworm, and Diatraea saccharalis (Fabricius), the Sugarcane Borer, respectively. The protocol described here can be used in similar species and includes (a) direct oral delivery by droplets containing dsRNA; (b) oral delivery by tomato leaflets that absorbed dsRNA solution; (c) delivery by Escherichia coli expressing dsRNA; and (d) delivery by transgenic plants expressing dsRNA.

Gene silencing by RNAi via oral delivery of dsRNA by bacteria in the South American tomato pinworm, Tuta absoluta.

BACKGROUND: RNA interference (RNAi) has been evaluated in several insect pests as a novel strategy to be included in integrated pest management. Lepidopterans are recognized to be recalcitrant to gene silencing by RNAi. As such, double-stranded RNA (dsRNA) delivery needs to be adjusted to assure its stability until it reaches the target gene transcript for silencing. Gene silencing by RNAi offers the potential to be used in the control of Tuta absoluta (Meyrick), one of the main insect pests of tomato (Solanum lycopersicum) worldwide. Here, we tested the delivery of dsRNA expressed in Escherichia coli HT115(DE3) and supplied to larvae in an artificial diet by screening target genes for silencing. We tested six target genes: juvenile hormone inducible protein (JHP); juvenile hormone epoxide hydrolase protein (JHEH); ecdysteroid 25-hydroxylase (PHM); chitin synthase A (CHI); carboxylesterase (COE); and arginine kinase (AK). RESULTS: Based on larval mortality, the duration of the larval stage in days, pupal weight, and the accumulation of the target gene transcript, we demonstrated the efficacy of bacterial dsRNA delivery for the functional effects on larval development. Providing dsRNA targeted to JHP, CHI, COE and AK by bacteria led to a significant decrease in transcript accumulation and an increase in larval mortality. CONCLUSION: Bacteria expressing dsRNA targeting essential T. absoluta genes supplied in artificial diet are efficient to screen RNAi target-genes. The oral delivery of dsRNA by bacteria is a novel potential alternative for the control of T. absoluta based on RNAi. © 2019 Society of Chemical Industry.

Investigating Engineered Ribonucleoprotein Particles to Improve Oral RNAi Delivery in Crop Insect Pests.

Genetically modified (GM) crops producing double-stranded RNAs (dsRNAs) are being investigated largely as an RNA interference (RNAi)-based resistance strategy against crop insect pests. However, limitations of this strategy include the sensitivity of dsRNA to insect gut nucleases and its poor insect cell membrane penetration. Working with the insect pest cotton boll weevil (Anthonomus grandis), we showed that the chimeric protein PTD-DRBD (peptide transduction domain-dsRNA binding domain) combined with dsRNA forms a ribonucleoprotein particle (RNP) that improves the effectiveness of the RNAi mechanism in the insect. The RNP slows down nuclease activity, probably by masking the dsRNA. Furthermore, PTD-mediated internalization in insect gut cells is achieved within minutes after plasma membrane contact, limiting the exposure time of the RNPs to gut nucleases. Therefore, the RNP provides an approximately 2-fold increase in the efficiency of insect gene silencing upon oral delivery when compared to naked dsRNA. Taken together, these data demonstrate the role of engineered RNPs in improving dsRNA stability and cellular entry, representing a path toward the design of enhanced RNAi strategies in GM plants against crop insect pests.

RNA interference as a gene silencing tool to control Tuta absoluta in tomato (Solanum lycopersicum).

RNA interference (RNAi), a gene-silencing mechanism that involves providing double-stranded RNA molecules that match a specific target gene sequence, is now widely used in functional genetic studies. The potential application of RNAi-mediated control of agricultural insect pests has rapidly become evident. The production of transgenic plants expressing dsRNA molecules that target essential insect genes could provide a means of specific gene silencing in larvae that feed on these plants, resulting in larval phenotypes that range from loss of appetite to death. In this report, we show that the tomato leafminer ( Tuta absoluta ), a major threat to commercial tomato production, can be targeted by RNAi. We selected two target genes (Vacuolar ATPase-A and Arginine kinase) based on the RNAi response reported for these genes in other pest species. In view of the lack of an artificial diet for T. absoluta, we used two approaches to deliver dsRNA into tomato leaflets. The first approach was based on the uptake of dsRNA by leaflets and the second was based on "in planta-induced transient gene silencing" (PITGS), a well-established method for silencing plant genes, used here for the first time to deliver in planta-transcribed dsRNA to target insect genes. Tuta absoluta larvae that fed on leaves containing dsRNA of the target genes showed an ∼60% reduction in target gene transcript accumulation, an increase in larval mortality and less leaf damage. We then generated transgenic 'Micro-Tom' tomato plants that expressed hairpin sequences for both genes and observed a reduction in foliar damage by T. absoluta in these plants. Our results demonstrate the feasibility of RNAi as an alternative method for controlling this critical tomato pest.