Bt Research 2025, Vol.16, No.6, 234-241 http://microbescipublisher.com/index.php/bt 237 much more difficult for insects to develop resistance to multiple toxins simultaneously (Li et al., 2016). In fact, this approach has been adopted by many genetically modified crops and has achieved good results. Not only is the toxicity range wider, but insects also cannot evolve resistance mechanisms so easily when facing these crops, and the development of resistance has been significantly slowed down. 4.2 RNA interference (RNAi) to enhance Bt toxin efficacy If Bt toxins are a "direct strike", then RNA interference is like "dismantling the enemy's weapons" behind the scenes. This technology can silence some key genes in pests, such as those that control detoxification, transport and detoxification, or toxin recognition. In this way, toxins are more likely to take effect and the resistance of pests is weakened (Li et al., 2016). The application methods of RNAi are quite flexible. Some use genetic engineering to make plants produce interference fragments on their own, while others directly apply them locally. Importantly, it has high precision and little impact on non-target organisms, and can be regarded as an environmentally friendly and efficient resistance countermeasure. 4.3 Gene editing (e.g., CRISPR/Cas) targeting insect resistance-related genes Nowadays, when people talk about resistance, more and more people are beginning to pay attention to CRISPR. Although most of these gene editing technologies are still being tested in laboratories at present, their potential should not be ignored. The CRISPR/Cas system can precisely modify the genes of insects, such as directly disrupting those key pathways or proteins that make them "immune" to Bt toxins (Li et al., 2016). If these resistance genes can be "invalidated", the insecticidal effect of Bt toxin may be enhanced again. Of course, there are still many problems to be solved for this technology to be widely applied in the fields. However, as an innovative means to supplement the existing Bt control system, its prospects are still worth looking forward to. 5 Development of Next-Generation Bt Toxins and Alternative Proteins 5.1 Screening and application of novel Bt toxin families (e.g., Vip, Cry51) Traditional Cry protein is not a panacea, especially when it comes to those resistant pests that have already been "trained". So, scientists began to pay attention to another batch of "players" - new Bt toxins like Vip and Cry51, whose structures and insecticidal methods are different from those of the older generation of Cry toxins. For instance, Vip protein is produced during the vegetive growth period of Bacillus thuriensis, which is different from the classic secretion mechanism of Cry and does not act on the same receptor. This makes it particularly effective in dealing with CR-resistant pests (Gupta et al., 2021). Especially Vip3, which has a very obvious effect on lepidoptera insects, has been combined with Cry protein into genetically modified crops such as cotton, corn and rice (Figure 2). Although these new toxins are not "substitutes", as supplementary measures, they do broaden the thinking of pest control and add an extra line of defense to the issue of resistance. 5.2 Synthetic Bt toxins and innovations in directed evolution Sometimes, ready-made Bt toxins are not effective, especially when dealing with pests that have evolved resistance. The original "routine" no longer works. At this point, some "surgery" is needed, which relies on protein engineering. Classic toxins like Cry1Ac, after undergoing directed evolution treatment, have their receptor recognition range broadened, allowing them to "see" targets that were previously unrecognized, and their insecticidal effect has also been enhanced (Badran et al., 2016). The principle behind this type of technology is actually not complicated. To put it simply, it is to "modify and upgrade" the old toxins to make them more suitable for the new resistance state of pests. Not only phage-assisted continuous evolution, but also screening methods such as phage display and ribosome display have gradually become routine operations in laboratories. They can quickly identify the best-performing ones among a bunch of toxin variants (Pacheco et al., 2015). Of course, in the final analysis, these methods are still a bit far from being widely used in the fields at present. But at least in terms of tool reserves, the "Arsenal" of Bt toxins is becoming increasingly flexible and targeted. 5.3 Development of non-Bt bioinsecticidal proteins (e.g., lectins, plant-derived proteins) If one keeps revolving around Bt, it is inevitable to encounter a bottleneck. So researchers have also begun to explore alternative approaches, attempting to find new insecticidal proteins from plants or other microorganisms.
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