The Scientist

An international team of researchers has developed a new technique for increasing pest resistance in transgenic crop plants, they report this week in PNAS. The strategy, which boosts and broadens the activity of Bacillus thuringiensis (Bt) toxins, targets previously impervious pest species and reduces by up to 1000-fold the level of toxin expression needed, said coauthor Paul Christou at the University of Lleida in Spain.

Heavy use of Bt insecticides worldwide has raised concern that insects might evolve resistance to Bt crops. Strategies to avoid the evolution of resistance include expressing multiple Bt toxins at high doses or fusing Bt toxins together, with resistance in both approaches requiring the unlikely acquisition of multiple simultaneous mutations. Christou and colleagues in Zimbabwe, Uganda, and Britain instead devised a new strategy that increases the repertoire of toxin-binding sites a Bt toxin attacks.

They fused the sequence for Bt toxin Cry1Ac with that of the nontoxic B-chain subunit of ricin (RB) in a recombinant plasmid. RB is a leptin that binds with galactose- and N-acetylgalactosamine residues with high affinity, the latter of which are key components of Bt toxin–binding receptors. They then bombarded embryonic callus from mature maize seeds with this BtRB fusion.

"The Bt toxins currently in crops have a very narrow host specificity, which is good because it reduces negative effects on beneficial insects and other nontarget organisms, including people, but it limits the application of any particular Bt toxin to controlling relatively few pests. So when you combine Bt toxins together, you're limited to the host specificities of each toxin," said Bruce Tabashnik at the University of Arizona in Tucson, who did not participate in this study. "With this novel technique, you don't have that limitation—you're broadening the specificity of an existing toxin by modifying its binding domain, rather than mixing and matching."

The researchers tested their fusion toxin against stem borer Chilo suppressalis, a pest normally susceptible to Cry1Ac, and found that maize producing low levels of BtRB killed 75% of larvae, compared with 17% in Bt-only plants. Similar trials with the cotton leaf worm Spodoptera littoralis, which is resistant to Bt delta endotoxins, showed that after 4 days, nearly 78% of larvae died on BtRB maize, compared to less than 20% on Bt-only or nontransformed maize. In the leafhopper Cicadulina mbila, which like other homopterans is ordinarily unaffected by Bt toxins, 95% of insects died by day 4 on BtRB maize, compared to 80% survival otherwise. In tests with the homopteran cereal aphid Rhopalosiphum padi, however, no toxicity was seen with BtRB or Bt-only maize.

Broadening the spectrum of species that Bt toxins attack could help control a wider range of pests, but "the potential negative effect is that you could increase toxicity to nontarget species such as humans or beneficial insects," Tabashnik told The Scientist.

Christou said his group was engaged in large-scale, collaborative experiments to determine exactly which insects are susceptible to these novel fusion proteins. "These results need to be validated repeatedly in multiple experiments, first in the laboratory, then in greenhouse experiments, and ultimately in controlled field experiments," he said. "If any of these experiments indicate toxicity to nontarget or beneficial insects, this toxicity needs to be studied, understood, and remedied before experiments progress forward."

Richard ffrench-Constant at the University of Bath in England, who did not participate in this study, cautioned that the increased toxicity of the fusion protein might instead be due to RB improving membrane insertion. "I'd like to see a Scatchard Plot of the binding of this fusion protein to brush border membrane preparations of different insect pests in order to confirm that activity against an increased range of insects is associated with altered binding," he told The Scientist.