Establishment of test systems for evaluating ingestion RNA interference-induced lethality against Tetranychus urticae, a representative sucking pest

Abstract

Double-stranded RNA-mediated interference (RNAi) has been universally applied as a specific, potent and successful technology for gene manipulation and emerged as a potential pest control strategy when combined with the plant transgenesis. To establish the RNAi-based control strategy against the two spotted-spider mite (Tetranychus urticae Koch), a worldwide notorious sucking pests in facility cultivation, test systems for evaluating ingestion RNA interference-induced lethality were established. As a prescreening tool prior to the development of transgenic crops, protocols for the agroinfiltration in the soybean and kidney bean were established and optimized. The maximum efficiency of hairpin RNA delivery in soybean was determined to be obtained by sea sand and syringe in the soybean and kidney bean, respectively. Transient expression of target hairpin RNA reached the maximum level at 24-h post-agroinfiltration in both soybean and kidney bean. The agroinfiltrated soybean plants expressing the COPA and aquaporin 9 (AQ9) hairpin RNA induced 64.1% and 39.5% mortalities of T. urticae at 144-h post-infestation. The target gene knocksown was observed in the infested mites, confirming that the observed mortality was likely resulted from RNAi. In summary, agroinfiltration was determined as an effective pre-screening tool for evaluating the RNAi-induced lethality of plants expressing target hairpin RNA prior to the generation of transgenic plants. To confirm the proof of concept that the constitutively expressed hairpin RNA in plant can also cause RNAi-induced lethality against T. urticae, a representative sucking pest, an Arabidopsis plant lines constitutively expressing the COPA hairpin RNA were generated via floral-dip method. When infested with T. urticae, the transgenic Arabidopsis resulted in T. urticae mortality and transcript downregulation. These findings strongly demonstrated that sap-feeding T. urticae can be actually killed by in planta expression of target lethal gene, such as COPA, and RNAi-based transgenic plants can be exploited as a novel alterative mean to control sucking pests T. urticae, as well.