Development of an agroinfiltration-based transient hairpin RNA expression system in pak choi leaves (Brassica rapa ssp. chinensis) for RNA interference against Liriomyza sativae

Abstract

The vegetable leafminer (Liriomyza sativae) is a devastating invasive pest of many vegetable crops and horticultural plants worldwide, causing serious economic loss. Conventional control strategy against this pest mainly relies on the synthetic chemical pesticides, but widespread use of insecticides easily causes insecticide resistance development and is harmful to beneficial organisms and environment. In this context, a more environmentally friendly pest management strategy based on RNA interference (RNAi) has emerged as a powerful tool to control of insect pests. Here we report a successful oral RNAi in L. sativae after feeding on pak choi (Brassica rapa ssp. chinensis) that transiently express hairpin RNAs targeting vital genes in this pest. First, potentially lethal genes are identified by searching an L. sativae transcriptome for orthologs of the widely used V-ATPase A and actin genes, then expression levels are assessed during different life stages and in different adult tissues. Interestingly, the highest expression levels for V-ATPase A are observed in the adult heads (males and females) and for actin in the abdomens of adult females. We also assessed expression patterns of the target hairpin RNAs in pak choi leaves and found that they reach peak levels 72 h post agroinfiltration. RNAi-mediated knockdown of each target was then assessed by letting adult L. sativae feed on agroinfiltrated pak choi leaves. Relative transcript levels of each target gene exhibit significant reductions over the feeding time, and adversely affect survival of adult L. sativae at 24 h post infestation in genetically unmodified pak choi plants. These results demonstrate that the agroinfiltration-mediated RNAi system has potential for advancing innovative environmentally safe pest management strategies for the control of leaf-mining species.

Keywords: Actin; Agroinfiltration-mediated RNAi; Liriomyza sativae; Pest control; V-ATPase a.