RNA interference in the Colorado potato beetle, Leptinotarsa decemlineata: Identification of key contributors

Abstract

RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. RNAi works very well in coleopteran insects including the Colorado potato beetle (CPB), Leptinotarsa decemlineata. We used a cell line (Lepd-SL1) developed from CPB to identify genes that play key roles in RNAi. We screened 50 genes with potential functions in RNAi by exposing Lepd-SL1 cells to dsRNA targeting one of the potential RNAi pathway genes followed by incubation with dsRNA targeting inhibitor of apoptosis (IAP, silencing of this gene induces apoptosis). Out of 50 genes tested, silencing of 29 genes showed an effect on RNAi. Silencing of five genes (Argonaute-1, Argonaute-2a, Argonaute-2b, Aubergine and V-ATPase 16 kDa subunit 1, Vha16) blocked RNAi suggesting that these genes are essential for functioning of RNAi in Lepd-SL1 cells. Interestingly, Argonaute-1 and Aubergine which are known to function in miRNA and piRNA pathways respectively are also critical to siRNA pathway. Using P labeled dsRNA, we showed that these miRNA and piRNA Argonautes but not Argonaute-2 are required for processing of dsRNA to siRNA. Transfection of pIZT/V5 constructs containing these five genes into Sf9 cells (the cells where RNAi does not work well) showed that expression of all genes tested, except the Argonaute-2a, improved RNAi in these cells. Results from Vha16 gene silencing and bafilomycin-A1 treatment suggest that endosomal escape plays an important role in dsRNA-mediated RNAi in Lepd-SL1 cells.

Identification and comparison of key RNA interference machinery from western corn rootworm, fall armyworm, and southern green stink bug

“…Poor expression of r2d2 has previously been suggested as a potential explanation of the insensitivity of a lepidopteran ovarian cell line to dsRNA [53]. Furthermore, a previous direct comparison of the expression of several core RNAi components in immortalized coleopteran pupal and lepidopteran ovarian cell lines showed universally lower expression in the lepidopteran cells, which was proposed to partially explain the observed discrepancy in dsRNA sensitivity [52]. Results from the current study indicate that expression levels of RNAi genes may not be a consistent source of disparity in whole insects, though r2d2 does show lower expression relative to the other core machinery genes.…”

“…Homologues of Dme ago2 have been reported in several lepidopteran and hemipteran pest species [49–51], though a FAW ago2 homologue was not included in a previous list of RNAi pathway genes detected in Sf21 cells [32]. Interestingly, AGO1 has also been reported to contribute to the response of a coleopteran cell line to exogenous dsRNA [52]. Although exact genomic copy number was not determined in this study, complete absence of any one core RNAi component—including both AGOs—cannot explain the difference in eRNAi response observed between WCR, FAW, and SGSB.…”

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

“…Apart from degradation in the digestive system, other research has shown the importance of functional uptake mechanisms for dsRNA. In insects, the primary mode of cellular uptake of dsRNA appears to be scavenger receptor-mediated endocytosis (Cappelle et al, 2016;Ivashuta et al, 2015;Li et al, 2015;Saleh et al, 2006;Ulvila et al, 2006;Wynant et al, 2014;Xiao et al, 2015;Yoon et al, 2016). Interestingly, also in adults of S.…”

Implementing the sterile insect technique with RNA interference – a review

“…melanogaster , for example, siRNA complexes with Ago2, miRNA associates with Ago1 (Tomari et al., ), and piRNA with PIWI proteins (Vagin et al., ; Malone et al., ). Although it is generally accepted that Ago2 is required for RNAi in the insects, silencing of Ago1 in Leptinotarsa decemlineata Say cells does inhibit gene silencing (Yoon et al., ). Humans, on the other hand, express four Ago proteins, all of which bind siRNA.…”