Enhancement of Bacillus thuringiensis Cry1Ab and Cry1Fa Toxicity to Spodoptera frugiperda by Domain III Mutations Indicates There Are Two Limiting Steps in Toxicity as Defined by Receptor Binding and Protein Stability

Abstract

Cry1Ab and Cry1Fa toxins are environmentally safe insecticides that control important insect pests. is an important maize pest that shows low susceptibility to Cry1A toxins, in contrast to Cry1Fa, which is highly active against this pest and is used in transgenic maize for control. The β16 region from domain III of Cry1Ab has been shown to be involved in interactions with receptors such as alkaline phosphatase (ALP) or aminopeptidase (APN) in different lepidopteran insects. Alanine-scanning mutagenesis of amino acids of Cry1Ab β16 (STLRVN) revealed that certain β16 mutations, such as N514A, resulted in increased toxicity of Cry1Ab for without affecting the toxicity for other lepidopteran larvae, such as larvae. Exhaustive mutagenesis of N514 was performed, showing that the Cry1Ab N514F, N514H, N514K, N514L, N514Q, and N514S mutations increased the toxicity toward A corresponding mutation was constructed in Cry1Fa (N507A). Toxicity assays of wild-type and mutant toxins (Cry1Ab, Cry1AbN514A, Cry1AbN514F, Cry1Fa, and Cry1FaN507A) against four populations from Mexico and one from Brazil revealed that Cry1AbN514A and Cry1FaN507A consistently showed 3- to 18-fold increased toxicity against four of five populations. In contrast, Cry1AbN514F showed increased toxicity in only two of the populations analyzed. The mutants Cry1AbN514A and Cry1AbN514F showed greater stability to midgut protease treatment. In addition, binding analysis of the Cry1Ab mutants showed that the increased toxicity correlated with increased binding to brush border membrane vesicles and increased binding affinity for ALP, APN, and cadherin receptors. is the main maize pest in South and North America and also is an invasive pest in different African countries. However, it is poorly controlled by Cry1A toxins expressed in transgenic crops, which effectively control other lepidopteran pests. In contrast, maize expressing Cry1Fa is effective in the control of , although its effectiveness is being lost due to resistance evolution. Some of the Cry1Ab domain III mutants characterized here show enhanced toxicity for without loss of toxicity to Thus, these Cry1Ab mutants could provide useful engineered toxins that, along with other Cry toxins, would be useful for developing transgenic maize expressing stacked proteins for the effective control of and other lepidopteran pests in the field.

Insect Hsp90 Chaperone Assists Bacillus thuringiensis Cry Toxicity by Enhancing Protoxin Binding to the Receptor and by Protecting Protoxin from Gut Protease Degradation

“…Recent work showed that S. frugiperda is less sensitive to Cry1Ab since Cry1Ab protoxin is highly unstable against S. frugiperda gut proteases and also because Cry1Ab showed a low binding affinity for the receptors located in the brush border membrane (BBM) of S. frugiperda midgut cells (39). Here, we show that PxHsp90 also increased Cry1Ab and Cry1Ac toxicity to S. frugiperda .…”

Making 3D-Cry Toxin Mutants: Much More Than a Tool of Understanding Toxins Mechanism of Action

“…Another area that has recently been demonstrated to be susceptible to improvement by site directed mutagenesis is the β sheet 16 in Domain III. A study of alanine substitution in Cry1Ab [97] performed on this area rendered mutants S509A, V513A, and N514A, which showed an increase in toxicity toward Spodoptera frugiperda of more than 9.5-, 12.7-, and 51-fold, respectively. As N514 was the most relevant position experiencing toxicity enhancement in β-16, saturation mutagenesis was performed at this position (N was changed for any of the other 19 amino acids).…”

The Cadherin Protein Is Not Involved in Susceptibility to Bacillus thuringiensis Cry1Ab or Cry1Fa Toxins in Spodoptera frugiperda

“…These results indicate that Cry1Ab toxicity is limited by some additional mechanisms, other than receptor binding, in the wild type DH19 larvae. It was reported that the lack of toxicity of Cry1Ab to an S. frugiperda population from México correlated with enhanced toxin degradation by midgut proteases and also with reduced receptor binding [32]. In addition, it is still possible that an additional Cry1Ab receptor is expressed in the Hi5 cells but not in S. frugiperda larvae.…”

“…In the case of T. ni, Cry1Ac variants that could bind to the TnCad protein were selected by continuous evolution, and it was found that the Cry1Ac variants that were able to bind to TnCad were also able to counter resistance of T. ni insects linked to ABCC2 mutations [40]. In addition, Cry1Ab domain III mutants were shown to increase the toxicity of Cry1Ab to different S. frugiperda strains, which was correlated with their increased binding to SfCad receptor [32]. Overall our results show that S. frugiperda cadherin is not a functional receptor of Cry1Fa and Cry1Ab toxins.…”

“…It has been shown that resistance to Cry1Fa in S. frugiperda is linked to different ABCC2 mutant alleles [27,28,31]. In addition, most S. frugiperda populations show low susceptibility to Cry1Ab or Cry1Ac toxins, in contrast to Cry1Fa that is highly active against this pest [21,32]. Here, we investigated whether SfCad is involved in the toxicity of Cry1Ab and Cry1Fa using both CRISPR/Cas 9 genome editing technology and cytotoxicity assays of Bt toxins in an insect cell line expressing SfCad.…”