Screening of lethal genes for feeding RNAi by leaf disc-mediated systematic delivery of dsRNA in Tetranychus urticae

Abstract

To identify genes that kill Tetranychus urticae when knocked down via RNA interference (RNAi), several lethal genes were screened by the systemic delivery of dsRNA via leaf disc feeding. Four candidate genes (β subunit of coatomer protein complex, T-COPB2; M1 metalloprotease, T-M1MP; Ribosomal protein S4, T-RPS4; A subunit of V-ATPase, T-VATPase) and a control gene (EGFP) were tested for RNAi. All dsRNAs that permeated the leaf disc (ca. 15-mm diameter) were detected at 12h post-treatment, indicating that dsRNA could move through vascular tissues. To evaluate RNAi toxicity, mortalities were assessed for 120h following treatment with dsRNA. Treatment with T-COPB2, T-M1MP, T-RPS4 and T-VATPase dsRNAs caused 65.4%, 15.9%, 36.1% and 21.1% mortalities at 120h post-treatment, respectively. Reduction of all target gene transcripts following dsRNA treatment was confirmed by quantitative PCR, demonstrating that dsRNA feeding-based RNAi could indeed kill T. urticae. In summary, dsRNA delivery via leaf disc is an effective system to screen for lethal genes. Furthermore, some genes, such as T-COPB2, T-M1MP, T-RPS4 and T-VATPase, can be used to establish an RNAi-based control system against T. urticae.