Researchers identify new pathway of resistance to one of the three insect-repelling proteins used in Bt cotton

Nearly 30 years ago, Australia’s first genetically modified field crop was planted: Bt cotton.

CSIRO scientists helped develop this insect-resistant cotton variety, using a gene derived from a soil bacteria, Bacillus thuringiensis (Bt).

Bt cotton works by producing a protein that kills the cotton bollworm—one of the crop’s major pests—when it feeds on its leaves.

It’s used by cotton growers in Australia and around the world, reducing the need for pesticides while increasing yields. It was first planted in 1996 after six years of extensive field trials and regulatory approval.

Now, CSIRO scientists have discovered how some insect pests are able to survive the insect-resisting effects of Bt cotton. The paper is published on the pre-print server bioRxiv.

It opens the door to better field monitoring of insect pest resistance using genetic screening to keep track of this form of genetic resistance, so Bt cotton can continue to be used successfully.

Cottoning onto pest resistance

But how do insect pests become resistant to these bacterial-based proteins?

CSIRO researcher with the Insects as Engineers team Dr. Andy Bachler wanted to find out.

He worked with Dr. Tom Walsh, CSIRO Applied Genomics Initiative co-lead, to unpick the threads of insect pest genomics.

“We knew some insects were showing resistance to the Vip3A protein in Bt cotton, and we had some clear data that this gene was doing something weird,” Andy said.

“But we couldn’t find the gene or figure out how this mode of resistance worked in global pest cotton bollworm (Helicoverpa armigera) or the Australian local species (Helicoverpa punctigera).”

Andy and Tom turned to long-read genomic sequencing to take a closer look at the pests.

Gene to be believed

“The long reads showed the gene and the mutation really clearly for the first time, whereas we couldn’t really see it with short reads,” Tom said.

It turns out the same key gene was disrupted in two different ways in separate groups of moths studied.

“There are two different types of mutation that give these cotton bollworms resistance. One is an insertion, and the other is a deletion,” Andy said.

The long-read sequencing showed there was a large insertion in a hidden part of the gene called the intron. The insertion was about 1½ times larger than the gene itself.

“The gene has been disrupted by something going into the intron, which normally you don’t really look at,” Andy said.

“It’s effectively invisible with the short reads, but when you use the right sequencing technology, you can see it,” Tom said.

Transposable elements—also called jumping genes—can jump around within the genome in various places in various ways. It’s part of the naturally occurring genetic mutations seen as species evolve and develop resistance to toxins.

Sometimes they jump into a gene and this disrupts the gene’s function.

Reaping what you sew

In the other type of genetic mutation discovered, there is a small deletion which means the gene doesn’t work anymore.

“There’s a deletion of a really important part of the gene which brings together a lot of the enzymes required to make transcription work,” Andy said.

“We don’t really know why this deletion occurred—it may be just by chance,” he said.

“With this gene, if you knock it out in two different ways, you get resistance.”

Spinning at life

Andy said insects develop resistance as genes mutate all the time.

“It sounds wrong but ‘breaking’ genes is key way for these insects to become resistant to the Bt toxins,” Andy said.

So, thanks to some random genetic mutation in nature, an insect is born with this broken gene.

The broken gene means the insect isn’t affected by the Bt toxin anymore. It’s a huge benefit, but, on the flipside, the broken gene also makes the insect a little less fit.

The gene is recessive, so the insect needs two broken copies to develop resistance.

“The adoption and the spread of these types of alleles into populations is essentially evolution in action,” Tom said.

“There’s a whole piece of work to be done now on exactly what this gene does and what role it plays.”

Staying a-thread of the curve

The identification of the resistance-specific gene is important for molecular pest monitoring in the field.

“Now we’ve found the gene we can keep an eye out for this in the field,” Andy said.

“It just gives us an extra place to look and another mechanism that we now know about.”

For now, this form of resistance in the field in Australia remains low and isn’t widespread.

“It means that all the crop management measures, including insect resistance management, are still working,” Andy said.

But globally resistance is appearing in the field in places like Brazil, China and America.

“If we see allele frequencies going up then we start to think we may have a problem,” Tom said.

“It could mean that either our resistance management is not working as well as it did before or that we have something coming in from overseas as a biosecurity issue.”

Molecular testing is again the key to monitoring. Long-read sequencing will be crucial for identifying resistance alleles in natural populations.

Remains to be gene

“The resistance gene that we have in this species, Helicoverpa, is relevant to a whole bunch of other pest species in the same way,” Tom said.

This gene is widespread across other insects. And it operates independently of other known resistance genes. So, it’s a new mechanism of Vip3A resistance.

CSIRO collaborator Professor Yidong Wu, from Nanjing Agricultural University, has found this same mechanism of resistance at work in the Fall Armyworm, another plant pest on the rise globally.

Ultimately, understanding this mechanism of resistance could open up further options for counteracting resistance or even help avoid the development of resistance.