Long dsRNAbut not siRNAinitiatesRNAi in western corn rootworm larvae and adults

Abstract

AbstractTransgenic maize plants expressing dsRNAtargeting western corn rootworm (WCR,Diabrotica virgifera virgiferaLeConte) v‐ATPase subunit CmRNAforRNAi provided significant root protection fromWCRlarval feeding damage in greenhouse assays compared to negative controls. Transcribed hairpin dsRNAinWCR‐resistant maize plants was present as both intact hairpin‐derived dsRNAand plant‐processed siRNA. Therefore, the ability of dsRNAand siRNAtargetingDv v‐ATPase CmRNAto cause anRNAi response was studied in bothWCRlarvae and adults. In 9‐day diet‐based feeding assays, dsRNAof at least 60 bp in length resulted in high levels of larval mortality. In contrast, 15‐, 25‐ or 27‐bp dsRNAs or pooled 21‐bp siRNAs did not cause mortality of exposed larvae. When larvae were fed with diet overlaid with siRNAs,Dv v‐ATPase Ctranscript levels did not change. Conversely, whenWCRlarvae were fed with diet overlaid with 184‐bp dsRNA, themRNAlevel was reduced by >20‐fold relative toyfpdsRNAnegative control. Similarly, 184‐bp dsRNAcaused 100% mortality ofWCRadults, whereas the mortality of adults fed on diet treated with siRNAs was similar to the negative control. Feeding adults with siRNAs on diet did not affect the level ofDv v‐ATPase CmRNAtranscripts, whereas adults fed with the 184‐bp dsRNAshowed approximately 35‐fold reduction in the targetmRNAlevel. Similar results were obtained with theWCRadults injected with 184‐bp dsRNAor 21‐bp siRNA. These results suggest that only long dsRNAor hairpin‐derived dsRNAis effective in causing lethal knock‐down ofDv v‐ATPase CmRNA. These results have implications for efficacious plant‐delivered dsRNAfor the protection of transgenic maize fromWCRfeeding damage and for the risk assessment of transgenic maize expressing insecticidal dsRNA.