## 3 RNAi Traits for Rootworm Control
### Target Site Discovery
#### 3.2.1 Larvicidal RNAi
Transgenic maize events expressing hairpin dsRNA targeting _v-ATPase A_ conferred root protection against WCR feeding damage, demonstrating that artificial diet-based mortality could translate to transgenic plant-based reduction in root damage. Further work on targets described by [PERSON] et al. [55] led to the commercial development of dsRNA maize events targeting WCR _sntf7_[124; 125; 135]. _Dv5 nf7_ is a WCR ortholog of the _Drosophila vps32_ or _shrub_, an essential component of the Endosomal Sorting Complex Required for Transport (ESCRT) pathway involved in intracellular protein trafficking [136]. Ultrastructural and histological studies showed that _sntf7_ suppression via exposure to _Dvsn7_/ dsRNA in diet feeding assays led to progressive degeneration of midgut enterocytes, cell sloughing, cell lysis, and larval mortality [125; 125]. RNAi-derived suppression of _sntf7_ represents the first RNAi-based MOA trait commercialized for insect pest control [32]. The successful engineering of _v-ATPase A_ dsRNA for _in planta_ control of WCR inspired RNAi target discovery research in WCR. Genome-wide screens utilizing high throughput target interrogation and knowledge-based approaches identified new RNAi-sensitive targets resulting in efficacious dsRNA maize events. [PERSON] et al. [123] tested 50 WCR genes selected based on genome-wide RNAi screens in _T. castaneum_[137]. From these, T0 maize plants expressing RNA hairpins for WCR orthologues _rop_ (vesicular trafficking), _dre4_ (transcription), or _rpIII140_ (transcription) showed protection from larval feeding damage. Knowledge-based approaches to dsRNA target selection have also been successful. [PERSON] et al. [114] hypothesized, based on information from _Drosophila_ genetic screens, that genes involved in insect midgut cell to cell septate junctions might be important to midgut integrity and function. Experimental evidence has shown that a class of WCR orthologous smooth septate junction (_ssj_) genes were sensitive to RNAi, resulting in insect mortality and plant protection. _DvSSJ1_, a homolog of the _snakeskin (ssk)_ gene from _Drosophila_, prevents luminal content leakage across the midgut epithelial lining. Ultrastructural and histochemical studies performed by [PERSON] et al. [114] showed that suppression of _DvSSJ1_ destroys the integrity of SSJs and results in the collapse of midgut epithelial cells into the lumen, which disrupts midgut function, leading to feeding cessation and subsequent larval mortality [114, 128]. Mortality directly correlated with a reduction in expression of _DvSSJ1_ transcript and protein in WCR larvae. [PERSON] et al. [127] examined a potentially haplolethal gene target, wings up A (_wup_A), which encodes Troponin I, an inhibitory protein of the Troponin-Tropomyosin complex involved in muscle contraction [138]. Due to the haplolethal phenotype, it was thought that RNAi of _wup_A might lead to greater dose sensitivity to dsRNA. RNAi of _wup_A resulted in a rapid onset of growth inhibition within two days. Knockdown of _wupA_ resulted in significant food accumulation in the hindgut due to a loss of peristaltic motion of the alimentary canal. Finally, [PERSON] et al. [126] examined the lethal impact of dsRNA targeting _sec23_, a coatomer protein, and a component of the (COPII) complex that mediates ER-Golgi transport [139]. _sec23_ was sensitive to RNAi in both larvae and adults. Interestingly, this study showed that 85% mRNA transcript knockdown resulted in 40% Sec23 protein knockdown, indicating that complete protein knockdown is not necessary to achieve insect mortality. Thus, it can be hypothesized that targeting important components of essential protein complexes could provide an effective route to insect control by disrupting protein complex stoichiometry.