## 3 RNAi Traits for Rootworm Control
### Target Site Discovery  
#### 3.2.1 Larvicidal RNAi  
Given the successful deployment of transgenic rootworm control traits to date, a primary focus for developing RNAi as another mode of action (MOA) for WCR control has been directed at identification and validation of genes where suppression of gene expression leads to rapid cessation of larval feeding, stunting, and lethality. The aspiration is to develop an RNAi-based trait with performance comparable to existing protein-based traits  
\begin{table}
\begin{tabular}{l l l l} \hline \hline \multicolumn{1}{c}{**Name**} & \multicolumn{1}{c}{**NCBI Accession No**} & \multicolumn{1}{c}{**Type of Exposure**} & \multicolumn{1}{c}{**Reference**} \\ \hline \multicolumn{4}{l}{**Larvicidal Genes**} \\ \hline _ESCRT III snf7_1  & XM\_028287710.1 & Artificial diet, _in planta_ & [31; 55; 124; 125] \\ _v-ATPase-A_1  & XM\_028281990 & Artificial diet, _in planta_ & [55] \\ _Actin_2  & XM\_028292745.1 & Artificial diet & [55] \\ _apple ATPase_1  & XM\_028281191.1 & Artificial diet & [55] \\ _alpha tubulin_1  & XM\_028282553.1 & Artificial diet & [55] \\ _beta tubulin_1  & XM\_028282553.1 & Artificial diet & [55] \\ _COPI customer subunit_\(\beta\)1  & XM\_02829120.1 & Artificial diet & [55] \\ _ESCRT III\_zps2_1  & XM\_02829669.1 & Artificial diet & [55] \\ _ESCRT I-Vps28_1  & XM\_02828797.1 & Artificial diet & [55] \\ _mvo_34  & XM\_028287237.1 & Artificial diet & [55] \\ _ribosomal protein L9_1  & XM\_028294395.1 & Artificial diet & [55] \\ _ribosomal protein L19_1  & XM\_028289442.1 & Artificial diet & [55] \\ _ribosomal protein S4_1  & XM\_028298505.1 & Artificial diet & [55] \\ _ribosomal protein trypsin-14_2  & XM\_028275245.1 & Artificial diet & [55] \\ _RNA polymerase II_1  & XM\_028297193.1 & Artificial diet & [55] \\ _vATPase-D_1 [FOOTNOTE:1]Footnote 1: Orthologes identified from _D. melanogaster_, 2 Orthologes identified from _D. in overall lethality and speed of kill. An RNAi-based MOA may result in overall mortality comparable to an insecticidal protein; however, generally, the kill speed is slower due to the intrinsic multistep cascade process that leads to gene suppression by RNAi [35; 133; 134; 55]. Consequently, selecting target gene(s) is an essential factor determining whether an RNAi-based MOA can be successfully developed as a control trait. In WCR, the RNAi targets do not have to be limited to the insect midgut since WCR mounts a strong systemic response against dsRNA [56; 113]. Ideal targets are those that are involved in critical physiological processes and, therefore, are essential to insect survival. Gene targets that have been characterized as effective candidates for larvicidal RNAi suppress essential biological functions including protein trafficking/sorting and transport (_sntf7_, _sec23_[126; 31]), organ integrity (_ssj1_[114]), energy metabolism (_v-ATPase_[55]), and muscle function (_troponin I_[127]) (Figure 1b). [PERSON] et al. [55] conducted the first large-scale screen of RNAi targets in WCR. A total of 290 dsRNA molecules were provided to WCR neonate larvae in diet feeding assays and numerous gene targets exhibited significant levels of mortality or growth inhibition. Among the most effective targets were putative orthologues for _vacuolar ATPase subunits A_ (_v-ATPase A_) and \(D\) (_v-ATPase D_), _COPI coatomer subunit_\(\beta\), ribosomal proteins, and _ESCRT III surf7_. Ingestion of dsRNA targeting mRNA for these and other genes resulted in various levels of larval stunting and mortality. For _v-ATPase A_, a corresponding suppression of mRNA levels and generation of homologous siRNAs were demonstrated. v-ATPases are ATP-dependent proton pumps that function to transport protons across plasma membranes, acidify intracellular compartments, and play an essential role in membrane trafficking and protein degradation [55]. Significantly, the work by [PERSON] et al. [55] provided the first demonstration of applying dsRNA technology for _in planta_ insect resistance.