Intensive genomic and transcriptomic research in various organisms in recent decades have identified a new class of RNAs called non-coding RNAs (ncRNAs). ncRNAs were initially considered as “junk” RNA molecules since they had no role in protein synthesis.Rigorous studies in this field have revealed that they play crucial roles in gene regulation, genome stabilization and defense against invading pathogens. As part of the RNA interference (RNAi) response, small ncRNAs between 18-36 nucleotides can be classified into microRNAs (miRNAs), short interfering RNAs (siRNAs), and PIWI-interacting RNAs (piRNAs) according to their biogenesis and function.
In this study, we attempted per oz administration of synthetic miRNA mimics and inhibitors to larvae to study the function of miRNAs. For this, we identified a miRNA, har-miR-2002b,that is specifically expressed during larval stages of Helicoverpa armigera (cotton bollworm) and used it as an example. The supply of har-miR-2002b by feeding larvae on the miRNA mimic led to 40% larval mortality, reduced adult emergence, and 70% reduction in adult fecundity. A potential target for this miRNA was identified as the H.armigera trypsin-like serine protease (Ha-TLP). The oversupply of har-miR-2002b significantly reduced the Ha-TLP’s transcript and protein levels leading to less trypsin activity in the midgut. On the other hand, the application of har-miR-2002b inhibitor increased the Ha-TLP’s transcript levels both in vitro and in vivo. We also confirmed this negative interaction using a GFP reporter study. This is the first study which showed the potential of utilization of miRNA mimics and inhibitors in insect miRNA research by oral feeding and their potential use in pest control by targeting specific insect genes.
By utilizing the per oz administration of miRNA mimics/inhibitors developed during this project, we successfully established that Helicoverpa armigera single nucleopolyhedrovirus (HaSNPV) down-regulates the host gene H. armigera ecdysone receptor (Ha-EcR) but not the control gene Ha-TLP, via down-regulating miR-14 around 72 hours post-infection. In vivo and in vitro studies showed that in the presence of the synthetic miR-14 mimic the transcript levels of Ha-EcR significantly increased. This positive interaction was further confirmed by a GFP reporter assay. Further, application of the miRNA mimic led to reduced replication of the virus. These data suggest that by downregulating miR-14 HaSNPV reduces the Ha-EcR transcript levels for its own benefit.
In our next attempt to identify potential virus-encoded miRNAs upon HaSNPV infection in H. armigera, we discovered the generation of viral siRNAs (v-siRNAs), as a consequence of RNAi response by H. armigera. We determined that the RNAi response was mediated mainly by Dicer-2 but not Dicer-1. In addition, the v-siRNAs did not uniformly map to the viral genome but rather found in several hotspots. In Dicer-2 silenced HzFB cells, we found higher transcript levels of hotspot genes and as a consequence more viral replication. Further, we demonstrated that the transcripts of key viral structural and auxiliary genes were the main substrates for Dicer-2 to generate v-siRNAs. The results clearly provided the first demonstration of the presence of an active RNAi response against a DNA virus. However, the virus may take advantage of this response to regulate its replication to avoid premature death of its host. Overall, the results of this project provide additional information demonstrating the significant role of snRNAs in host-pathogen interaction.