Bacillus thuringiensis (Bt) crops have been one of the most successful applications of genetic engineering in agriculture. The crops carry a gene that encodes a bacterial protein that kills insects that ingest it. While it's possible to spray crops with the Bt toxin instead, farms that rely on Bt GMO crops are more profitable, have higher productivity, and use less pesticides.

Unfortunately, evolution isn't sitting still, and Bt-resistant insects are beginning to become a problem. While scientists are developing new crops with other Bt genes and farmers can adopt agricultural practices that limit the risk of resistance, some researchers decided to short-circuit the whole process. In a new paper, they figured out how to evolve a completely new Bt toxin in a virus that infects bacteria and showed that it was effective in killing insects.

The basics of Bt

The researchers' work highlights the importance of basic, fundamental research—while a handful of the research team worked at Monsanto, two-thirds came from various academic institutions. And the work relies on some basic information that's not essential to using Bt in GMOs.

That information includes details like the mode of action of the toxin, which binds to proteins on the surface of cells in insect guts, opening pores that eventually kill the cells. The structure of the Bt protein has also been studied, allowing researchers to identify the parts of the protein that bind targets on the insect cells. We've also studied Bt-resistant insects and found that the resistance evolves by reducing or eliminating the expression of the protein that Bt binds to.

So, the researchers reasoned, the simplest way to avoid this resistance is to get the Bt protein to bind to something else on the cells of an insect's guts. Other research has already identified a large catalog of potential targets. As noted above, the structure of the Bt protein was available, so the researchers knew precisely which part of the protein had to be changed. The only question was how to actually re-engineer the targeting.

Rather than doing the re-engineering themselves, the researchers decided to let evolution do it for them. To get this idea to work, they essentially redesigned an experimental system that was originally intended to help identify when two proteins stuck to each other (biologists will recognize this as a variant of a two-hybrid system).

Evolution in action

They started with one of the proteins known to be expressed on the guts of insect cells (a cadherin-like receptor). Part of that gene—we'll call it the bait—was fused onto a DNA binding protein, allowing the whole thing to stick to a specific DNA sequence. That sequence was then placed next to a gene that's essential for the reproduction of a virus that targets bacteria. On its own, however, there's nothing about this hybrid DNA binding protein that would ensure the gene would get made into a protein. So a virus with only this setup wouldn't reproduce.

To get the virus to survive, the authors fused the targeting portion of the Bt protein—we'll call that the fish—with a viral protein that transcribes genes into messenger RNA. If the fish can interact with the bait, then any genes near where the bait binds to DNA will be made into proteins. If that happens, the essential virus gene gets made, allowing the virus to reproduce in bacteria.

That process required a tremendous amount of work to set up and test. But testing revealed that it didn't work. There was so little interaction between the fish and the bait that no viruses could survive. So the authors had to create a brand new fusion protein with a bait that was halfway between an existing Bt target protein and the new, intended target protein. This work finally got enough protein made for the virus to reproduce. It didn't reproduce efficiently, but it was enough for evolution to work with.

The authors then forced the virus to evolve for more than 500 hours. Once the first bait-fish system (the one that was halfway to the intended target) worked well, they switched the bait to the final target sequence and cranked up the rate of mutations. At the end of all that evolution, the authors sequenced all the fish proteins that worked efficiently. They found that evolution had produced a variety of solutions, some of them carrying up to 16 different mutations. Twenty-two different mutations were identified in the fish overall.

So the authors engineered a new version of the Bt toxin, one that carried the newly evolved portion of the fish domain. When they tested it on insect cells... it didn't work all that well. This result really shouldn't come as a surprise; the fish-bait interaction was selected for inside a bacterial cell, which is a very different environment from an insect gut. The authors suspect that the newly evolved portion of the protein was destabilizing it in the insect gut, causing the entire toxin to fall apart.

Try, try again

The researchers took their list of 22 mutations and started testing different combinations, eventually identifying two specific mutations that were responsible for destabilizing the toxin. (This is much more work than that last sentence would imply.) They tested a number of combinations with the remaining 20 mutations and found one that worked well.

The resulting protein successfully killed insect cells, and it also was effective against larva of the cabbage looper, a known agricultural pest. The authors also tested a strain of cabbage looper that was resistant to an existing Bt toxin. The newly evolved version of Bt killed them just as efficiently as it killed normal strains.

All of this was a staggering amount of work. But the authors argue it was worth it because the basic approach of bait-fish evolution can be re-deployed for a variety of purposes. Want to target a completely different type of insect? It should work. Want to target a completely different protein in a known pest? Shouldn't be a problem. With a large collection of new Bts, we should be able to handle the issue of existing Bt-resistant insects as well as engineer a new collection of crops that carry multiple Bts, making evolution of additional resistance unlikely.

And that's just the benefits in pest control. The authors note that similar approaches are already being attempted with medically relevant targets. Sometimes, biotech makes other technology fields look positively conservative.

Nature, 2016. DOI: 10.1038/nature17938 (About DOIs).