A look at resistance management in biotechnology.

Written by Joe Ballenger

I work in industry, so it’s not often that I can talk about what I do. The reason for this is that I work in a group called discovery, which means that I look for new proteins which we can use against crop pests. The majority of what I find doesn’t work, which is kind of par for the course. However, when we find something that only works against certain insects, we slap it in a plant and then wait a decade for it to come on the market after a whole host of safety tests. In agricultural biotechnology, our goal is to find things that are specific. That is, we want to use stuff that only targets insects…and specific groups of insects at that.

Double-stranded RNA is perfect for that. Critters use dsRNA to do a whole lot of things, from fighting infections to turning specific genes on and off while they’re growing up. It’s purpose is to be very specific, and only act on certain genes. It’s the perfect tool for agriculture, because if we find the right gene sequence, we can make a pesticide that is species specific.

Beyond that we also need things that are durable; that are resistant to evolution so they’ll be on the market for awhile. Some animals are so well known for what they do, that we can associate them with a certain lifestyle. Fish live in the water, so they swim. Birds get around by flying, so they’re associated with flight. Well, insects evolve resistance to everything we’ve ever used to control them. So birds fly, fish swim, and insects evolve.

dsRNA was supposed to be a perfect tool. It was species specific, safe for use in food plants. Hypothetically it was supposed to be durable, as well. This is a pretty important pathway, because bugs need it to fight diseases and work their genes. The idea was that if insects became resistant to one gene, that gene could be swapped out with something else pretty easily.

Well…even seemingly obvious hypothesis need to be tested, because nature doesn’t always work like we think it should.

A little bit of background…

WCR knockdown

A big deal is always made about the role of RNAi in the immune system, but it’s just as important for development. The deformed insect on the right was produced by knocking down the RNAi pathway, leading to a whole host of deformities which make the insects less healthy. Image credit: Davis-Vogel et. al 2018

The thing to know about dsRNA is that this is a completely new paradigm in insect control. It was discovered about 20 years ago, and folks have been working to see if it could be used ever since. The transgenic crops were the most obvious choice, but spray formulations are also something that’s been under development. In fact, making spray dsRNA formulations an economic reality have required the invention of a completely new type of bioreactor. The new crops, which have a dsRNA called DvSnf7 inserted into them, are target against Western Corn Rootworm…a pest that can cause billions of dollars in lost crops every year.

It was pretty quickly discovered that triggering the RNAi pathway is easier in some insects than others. Caterpillars, for instance, have enzymes which break down dsRNA before it hits it’s target. So do aphids. Lots of insects are resistant to dsRNA for one reason or another, and it seems like broad-spectrum dsRNA resistance is common despite it’s importance to the immune system and insect development.

Resistance

So it’s already reasonable to think that resistance could develop to dsRNA brought in from outside the bug, especially if it’s eaten. After all, all sorts of germs can get in through the mouth and it only makes sense to have some sort of defense system against viruses. Because plants also use dsRNA in their development, it also makes sense that you’d do something to keep your food from interfering with your machinery. Quite frankly, I’ve always been surprised that it actually worked…but it does and that’s the amazing thing.

Some of the folks I work with (I wasn’t directly involved with this research, by the way) bred some Western Corn Rootworm that was resistant to the dsRNA that’s going to be used in crops that were just approved by the USDA last month. This work all took place in the laboratory (which is important), and they tested this resistant colony against dsRNA to several well known genes.

dsRNA WCR resistance

The gene that’s being targeted in the crops is a gene called DvSnf7, and it attacks the pathway that lets cells take stuff up. COPI is a different gene in that process, while vATPase makes energy. Mov34 gets rid of un-needed proteins. While DvSnf7 is the important one for transgenic crops, the other proteins (vATPase and Mov34) are completely different genes involved in completely different pathways.

The insects which were bred to be resistant to DvSnf7 are resistant to dsRNA from other genes. If RNAi resistance was based on sequence, like many had thought it would be, the insects would have survived eating dsRNA with other genes.

How does the resistance work?

Here’s where things actually get kind of cool. See, we can actually take pictures of the dsRNA molecules. We can incubate the dsRNA with stomach extract from the insects, and use dye which binds to RNA to see if it’s getting degraded. We can also tag a red dye called Cy3 onto the dsRNA and see if the insect’s cells will let it in.

dvsnf7 resistance mechanism.png

In the image to the left, the blue dye tells us where the insect cells are by labeling the nucleus in a nice shade of blue. The dsRNA is labeled in red. When you combine these images, it tells you whether the dsRNA has been taken up by the cells.
The image to the left is important because it tells us two things. Unlike other bugs which destroy dsRNA before the cells take it up, the resistant Western Corn Rootworm takes a different tactic…it doesn’t even bother letting the dsRNA into the cells.
This is…kind of a problem. It’s not that these bugs are degrading the dsRNA like caterpillars or aphids, it’s that the dsRNA is going right through the bug like a bad taquito. The dsRNA doesn’t even have a chance to act, because it’s not being let into the insect in the first place.

Looking to the future

To combat dsRNA resistance, we need to understand why, how, and when insects will become resistant…or why they don’t become resistant to certain things. Increasingly, this research will be done alongside new things which come to market.

Western Corn Rootworm is kind of weird in that RNAi is set off so easily. They do vector viruses to different plants, but the viruses they’re known to vector don’t reproduce inside of them. Other important plant disease vectors, like aphids and thrips, have viruses which replicate inside of them.

I mention this, because we should highlight that this research was performed in a laboratory colony. Nobody knows whether these insects could survive in the wild, or what effect the lack of dsRNA uptake would have on the ability of the insects to fight disease. There aren’t a whole lot of viruses known to infect this particular species, so it’s hard to answer this question in the lab. It may be that these bugs constantly sample RNA from their environment to ward off disease, although we’d need to challenge these insects with pathogens to know that for sure.

For now, we should just be confident in saying that dsRNA inserted into crops will have to be managed very differently than other insect control measures like Bt proteins. It may be that for most insects, spray formulations will work better in the long term…and those sprays will have to be designed with encapsulating agents that let the dsRNA into the cells.

I don’t think we should be quite ready to throw the towel in on dsRNA quite yet. However, it’s a new technology and it will need to be developed more to keep it as viable for as long as possible.

Disclaimer:

Joe Ballenger works as a contractor for Monsanto, the company which developed and commercialized DvSnf7. He did not participate in the research.

Works Cited

Davis-Vogel, C., Ortiz, A., Procyk, L., Robeson, J., Kassa, A., Wang, Y., … & Sashital, D. G. (2018). Knockdown of RNA interference pathway genes impacts the fitness of western corn rootworm. Scientific Reports, 8(1), 7858.

Khajuria, C., Ivashuta, S., Wiggins, E., Flagel, L., Moar, W., Pleau, M., … & Hodge, T. (2018). Development and characterization of the first dsRNA-resistant insect population from western corn rootworm, Diabrotica virgifera virgifera LeConte. PloS one, 13(5), e0197059.

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