IPD072Aa from Pseudomonas chlororaphis is a new insecticidal protein that has been shown to have high activity against western corn rootworm (WCR). IPD072 has no sequence signatures or predicted structural motifs with any known protein revealing little insight into its mode of action using bioinformatic tools. As many bacterially derived insecticidal proteins are known to act through mechanisms that lead to death of midgut cells, we evaluated whether IPD072Aa also acts by targeting the cells of WCR midgut. IPD072Aa exhibits specific binding to brush border membrane vesicles (BBMVs) prepared from WCR guts. The binding was found to occur at binding sites that are different than those recognized by Cry3A or Cry34Ab1/Cry35Ab1, proteins expressed by current maize traits that target WCR. Using fluorescence confocal microscopy, immuno-detection of IPD072Aa in longitudinal sections from whole WCR larvae that were fed IPD072Aa revealed the association of the protein with the cells that line the gut. High-resolution scanning electron microscopy of similar whole larval sections revealed the disruption of the gut lining resulting from cell death caused by IPD072Aa exposure. These data show that the insecticidal activity of IPD072Aa results from specific targeting and killing of rootworm midgut cells. IMPORTANCE Transgenic traits targeting WCR based on insecticidal proteins from Bacillus thuringiensis have proven effective in protecting maize yield in North America. High adoption has led to WCR populations that are resistant to the trait proteins. Four proteins have been developed into commercial traits, but they represent only two modes of action due to cross-resistance among three. New proteins suited for trait development are needed. IPD072Aa, identified from the bacterium Pseudomonas chlororaphis, was shown to be effective in protecting transgenic maize against WCR. To be useful, IPD072Aa must work through binding to different receptors than those utilized by current traits to reduce risk of cross-resistance and understanding its mechanism of toxicity could aid in countering resistance development. Our results show that IPD072Aa binds to receptors in WCR gut that are different than those utilized by current commercial traits and its targeted killing of midgut cells results in larval death.
Keywords: Diabrotica virgifera virgifera; Pseudomonas chlororaphis; bacterial toxin; biotechnology; confocal microscopy; electron microscopy; insect; rootworm.