## 2 B. _thuringiensis_
### Vegetative Insecticidal Proteins  
The application of \(Bt\)-derived insecticidal proteins was rapidly adopted by farmers across the world after their introduction in the mid-1990s [23]. As of 2020, 82 percent of the total maize acres in the US were sown with \(Bt\) transgenic maize [4]. However, much like the pervasive use of antibiotics which has led to the emergence of antimicrobial resistance (AMR), so too has extensive genetic modification of crops led to the emergence of \(Bt\) toxin-resistant pests. Notorious among them is _Diabrotica virgifera virgifera_, the western corn rootworm (WCR), which has acquired resistance to Cry3 Bb1 and Gpp34 Ab1/Tpp35 Ab1 (formally known as Cry34 Ab1 and Cry35 Ab1) [4; 24]. This rise in transgenic crop resistance has motivated the search for new tools to combat crop infestations. These include Vpb1 and Vpa2 (formerly ViP1 and ViP2), which are toxic towards pests of the _Hemiptera_ (piercing-sucking True Bugs) and certain _Coleoptera_ species [16]. Vpb1 and Vpa2 work in concert as an AB-type binary toxin system, where Vpb1 binds to insecticide midgut receptors and forms an endosomal channel used by Vpa2 to enter the cytosol of target cells. Following this translocation, Vpa2 then ADP-ribosylates actin, leading to cytoskeletal disruption and lethal cytotoxicity [16]. Another promising Vip expressed by \(Bt\) is Vpb4 Da2, the first insecticidal Vpb4 protein exhibiting commercial-level control against the WCR [4; 24]. Vpb4 Da2 is composed of protective antigen 14 (PA14) and Binary-toxB protein family domains [4]. However, unlike the Vpb1/Vpa2 binary toxin system, Vpb4 Da2 does not recruit or require an enzymatic partner to facilitate toxicity [4]. This was observed upon feeding WCR larvae Vpb4 Da2 alone, which led to the blebbing of midgut epithelial cells six hours post-ingestion, the sloughing off of cellular debris in the WCR lumen, and the partial disruption of the apical microvilli layer 48 h post-ingestion, with nearly complete loss of the microvilli layer 96 h post-ingestion [4]. Fascinatingly, assessment of the amino acid sequence of Vpb4 Da2 revealed that an alanine is present in place of the phenylalanine found in other binary toxin B subunits (Figure 2) [25]. As discussed below (Section 5), this phenylalanine is highly conserved and critical for A subunit unfolding and translocation. These observations suggest Vpb4 Da2 is unlikely to serve as a transporter of a toxic payload due to the importance this phenylalanine plays in enzymatic A subunit linearization and translocation. This is further reinforced by the ability of Vpb4 Da2 to be highly toxic on its own [4]. Despite this, the mode of action for Vpb4 Da2 remains unclear. Regardless, Vpb4 Da2 shows structural homology to the PA from anthrax toxin and C2 II from \(B\). _ botulinum_ C2 toxin, qualifying its inclusion in this discussion of bacterial pore-forming binary toxins [4]. Furthermore, studies were performed to benchmark the safety of Vpb4 Da2, confirming that it is eventually degraded by gastrointestinal proteases in vitro and shows no signs of toxicity in CD-1 mice after oral challenge [25]. These results indicate that Vpb4 Da2 (and potentially other Vpb4 toxins) are promising additions to the current repertoire of \(Bt\)-derived toxins used to protect crops from insect-derived destruction. Figure 2: Comparative analysis of B subunits from select Clostridal and _Bacillus_ binary toxins. Sequences were retrieved from UniProt or NCBI databases and were aligned using MUSCLE (default parameters) with structural and biochemical data superimposed [26]. Cd, _Clostridioides difficile_ (CDTb: F5B5U8); Cp, _Clostridium perfingens_ (Ib: Q46221, BECb: XSHZK7); Ts, _Thomasclaveglia saccharomyces_ expression system (CSTb: WP_227053651); Cb, _Clostridium botulinum_ (C2 II: D4N872); Ba, _Bacillus anthrac_ (PA, P13423); and Bt, _Bacillus thuringiensis_ (Vpb4: A0A2U5 FTM5, Vpb1: M1 QYU2). Bracketed values indicate the number of aligned residues between key structural and functional features for the B subunits presented.