Specific binding between Bacillus thuringiensis Cry9Aa and Vip3Aa toxins synergizes their toxicity against Asiatic rice borer (Chilo suppressalis)

Abstract

The bacterium produces several insecticidal proteins, such as the crystal proteins (Cry) and the vegetative insecticidal proteins (Vip). In this work, we report that a specific interaction between two toxins creates insecticidal synergism and unravel the molecular basis of this interaction. When applied together, the three-domain Cry toxin Cry9Aa and the Vip Vip3Aa exhibited high insecticidal activity against an important insect pest, the Asiatic rice borer (). We found that these two proteins bind specifically to brush border membrane vesicles of and that they do not share binding sites because no binding competition was observed between them. Binding assays revealed that the Cry9Aa and Vip3Aa proteins interacted with high affinity. We mapped their specific interacting regions by analyzing binding of Cry9Aa to overlapping fragments of Vip3Aa and by analyzing binding of Vip3Aa to individual domains of Cry9Aa. Binding to peptide arrays helped narrow the binding sites to domain II loop-3 of Cry9Aa and toTKKMKTL in Vip3Aa. Site-directed mutagenesis confirmed that these binding regions participate in binding that directly correlates with the synergism between the two proteins. In summary, we show that the Cry9Aa and Vip3Aa toxins display potent synergy based on a specific interaction between them. Our results further our understanding of the complex synergistic activities among toxins and are highly relevant to the development of toxin combinations for effective insect control and for delaying development of insect resistance.

Current Insights on Vegetative Insecticidal Proteins (Vip) as Next Generation Pest Killers

“…Interestingly, the synergism between Vip3Aa and Cry9Aa mutants was disturbed moderately in C. suppressalis, and severely in and O. furnacalis. Synergism resulted in an improved toxicity of Vip3Aa and Cry9Aa in C. suppressalis, which is a great threat to rice crops in China [106]. Strong synergism was also seen in the Cry1Ab/Vip3Ca protein combination.…”

Insecticidal Activity and Synergistic Combinations of Ten Different Bt Toxins against Mythimna separata (Walker)

“…A number of investigations have confirmed that synergism and antagonism may occur between Vip3 and Cry proteins [ 57 , 58 , 59 , 60 ]. Bergamasco et al reported the species specificity effect of protein interaction between Cry1Ia and Vip3Aa [ 61 , 62 , 63 ]. A synergistic effect was found in FAW, Spodoptera albula , and Spodoptera cosmioides , but an antagonistic effect was found in Spodoptera eridania .…”

Structural and Functional Insights into the C-terminal Fragment of Insecticidal Vip3A Toxin of Bacillus thuringiensis

“…Among the Vip toxin family, Vip3A toxins are the most abundant and most studied [8]. Compared with known Cry toxins, Vip3A toxins share no sequence homology, bind to different receptors [10][11][12][13], and lack cross-resistance [14][15][16][17], therefore they are considered as ideal options to complement and expand the use of Bt in crop protection and resistance management. At present, the Vip3Aa toxin is the only family member that has been used in commercial transgenic crops together with Cry toxins, and no field-evolved resistance has yet been reported [1,8,18].…”