Indian scenario on the occurrence of a dreaded insect pest Pink bollworm, Pectinophora gossypiella on Bt cotton-A review

The pink bollworm, Pectinophora gossypiella, has become a significant production constraint on Bt cotton in India. This problem is unique to India because the pest has developed multi-fold resistance to Cry toxins in many Indian populations but not in other countries. Most Indian populations have developed multifold resistance to Cry 1 Ac and Cry 1Ac + Cry 2 Ab toxins. Year-round cultivation of long-duration Bt cotton hybrids on a large scale has a pronounced impact on the incidence. Also discussed other factors responsible for the occurrence of pink bollworm on Bt cotton in India. Insecticide Resistance Management (IRM) strategies implemented by different cotton-growing countries globally; the USA, India, and China had a significant impact on the interaction of pink bollworm on Bt cotton. Huge selection pressure resulted in resistance to Cry toxins. Time-tested IPM, if implemented on a community basis focusing on pheromones technology and closed season, will help sustain the cotton cultivation in India in the future. Thus, this review aims to congregate exhaustive information on the history, biology, resistance to Bt cotton, and Integrated Management (IPM) options for the Indian scenario, which would help researchers in their future endeavors.

Effect of Cry Toxins on Xylotrechus arvicola (Coleoptera: Cerambycidae) Larvae

The beetle Xylotrechus arvicola is a destructive pest in vineyards (Vitis vinifera) in the main wine-producing areas of the Iberian Peninsula. X. arvicola larvae bore into the grapevine wood-making galleries, thus damaging the plant both directly and indirectly; the latter through the proliferation of wood fungi, which can invade the inside of the plant, decreasing the quality and quantity of its production. The susceptibility of X. arvicola larvae to five coleopteran toxic Cry proteins (Cry1B, Cry1I, Cry3A, Cry7A, and Cry23/37) was evaluated under laboratory conditions in order to deepen the knowledge of the effect of these proteins on this insect throughout its biological development.

Key residues of Bacillus thuringiensis Cry2Ab for oligomerization and pore-formation activity

As a pore-forming toxin, activation, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous results revealed that the helices α4–α5 of Domain I were involved in the oligomerization of Cry2Ab, however, the key residues for Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site-directed in the helices α4–α5 of Domain I and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A could reduce the assembly of the 250 kDa oligomers, suggesting that these mutation residues might be essential for Cry2Ab oligomerization. As expected, all of these variants showed lower insecticidal activity against P. xylostella. Furthermore, we found that the pore-forming activities of these mutants also decreased when compared to wild-type Cry2Ab. Taken together, our data identified key residues for Cry2Ab oligomerization and emphasized that oligomerization was closely related to the insecticidal activity and pore-forming activity of Cry2Ab.

A cis-Acting Mutation in the PxABCG1 Promoter Is Associated with Cry1Ac Resistance in Plutella xylostella (L.)

The molecular mechanisms of insect resistance to Cry toxins generated from the bacterium Bacillus thuringiensis (Bt) urgently need to be elucidated to enable the improvement and sustainability of Bt-based products. Although downregulation of the expression of midgut receptor genes is a pivotal mechanism of insect resistance to Bt Cry toxins, the underlying transcriptional regulation of these genes remains elusive. Herein, we unraveled the regulatory mechanism of the downregulation of the ABC transporter gene PxABCG1 (also called Pxwhite), a functional midgut receptor of the Bt Cry1Ac toxin in Plutella xylostella. The PxABCG1 promoters of Cry1Ac-susceptible and Cry1Ac-resistant strains were cloned and analyzed, and they showed clear differences in activity. Subsequently, a dual-luciferase reporter assay, a yeast one-hybrid (Y1H) assay, and RNA interference (RNAi) experiments demonstrated that a cis-mutation in a binding site of the Hox transcription factor Antennapedia (Antp) decreased the promoter activity of the resistant strain and eliminated the binding and regulation of Antp, thereby enhancing the resistance of P. xylostella to the Cry1Ac toxin. These results advance our knowledge of the roles of cis- and trans-regulatory variations in the regulation of midgut Cry receptor genes and the evolution of Bt resistance, contributing to a more complete understanding of the Bt resistance mechanism.

Which Is Stronger? A Continuing Battle Between Cry Toxins and Insects

In this article, we review the latest works on the insecticidal mechanisms of Bacillus thuringiensis Cry toxins and the resistance mechanisms of insects against Cry toxins. Currently, there are two models of insecticidal mechanisms for Cry toxins, namely, the sequential binding model and the signaling pathway model. In the sequential binding model, Cry toxins are activated to bind to their cognate receptors in the mid-intestinal epithelial cell membrane, such as the glycophosphatidylinositol (GPI)-anchored aminopeptidases-N (APNs), alkaline phosphatases (ALPs), cadherins, and ABC transporters, to form pores that elicit cell lysis, while in the signaling pathway model, the activated Cry toxins first bind to the cadherin receptor, triggering an extensive cell signaling cascade to induce cell apoptosis. However, these two models cannot seem to fully describe the complexity of the insecticidal process of Cry toxins, and new models are required. Regarding the resistance mechanism against Cry toxins, the main method insects employed is to reduce the effective binding of Cry toxins to their cognate cell membrane receptors by gene mutations, or to reduce the expression levels of the corresponding receptors by trans-regulation. Moreover, the epigenetic mechanisms, host intestinal microbiota, and detoxification enzymes also play significant roles in the insects’ resistance against Cry toxins. Today, high-throughput sequencing technologies like transcriptomics, proteomics, and metagenomics are powerful weapons for studying the insecticidal mechanisms of Cry toxins and the resistance mechanisms of insects. We believe that this review shall shed some light on the interactions between Cry toxins and insects, which can further facilitate the development and utilization of Cry toxins.

Key Residues of Bacillus Thuringiensis Cry2Ab for Oligomerization and Pore-Formation Activity

As a pore-forming toxin, oligomerization and pore-formation were both required for the mode of action of Cry toxins. Previous studies revealed that Domain I helices α4-α5 were involved in oligomerization of Cry2Ab, while the active residues in charge of Cry2Ab aggregation remained ambiguous. In present studies, we built 20 Cry2Ab alanine mutants site directed in helices α4-α5 and demonstrated that mutants N151A, T152A, F157A, L183A, L185A and I188A blocked the assembly of 250 kDa oligomers, suggesting that those residues were key residues for Cry2Ab oligomerization. As expected, those variants severely reduced the insecticidal activity against P. xylostella which was similar to our previous reports. Furthermore, we found that the pore-forming activities of non-oligomerization mutants sharply decreased compared to wild-type Cry2Ab. Taken together, our data comprehensively identified key residues for Cry2Ab for the first time and emphasized that oligomerization was closely related to insecticidal activity and pore-forming activity in Cry2Ab.

The Essential and Enigmatic Role of ABC Transporters in Bt Resistance of Noctuids and Other Insect Pests of Agriculture

In the last ten years, ABC transporters have emerged as unexpected yet significant contributors to pest resistance to insecticidal pore-forming proteins from Bacillus thuringiensis (Bt). Evidence includes the presence of mutations in resistant insects, heterologous expression to probe interactions with the three-domain Cry toxins, and CRISPR/Cas9 knockouts. Yet the mechanisms by which ABC transporters facilitate pore formation remain obscure. The three major classes of Cry toxins used in agriculture have been found to target the three major classes of ABC transporters, which requires a mechanistic explanation. Many other families of bacterial pore-forming toxins exhibit conformational changes in their mode of action, which are not yet described for the Cry toxins. Three-dimensional structures of the relevant ABC transporters, the multimeric pore in the membrane, and other proteins that assist in the process are required to test the hypothesis that the ATP-switch mechanism provides a motive force that drives Cry toxins into the membrane. Knowledge of the mechanism of pore insertion will be required to combat the resistance that is now evolving in field populations of insects, including noctuids.