A Game-Changing Plant Health Technology
By Carol Miller, Editor, American Vegetable Grower
On Christmas Eve 2020, a group of scientists released a report on a revolutionary new method for battling COVID-19. As a couple of ag researchers read through the findings, they became more and more excited. The potential of the described technology seemed almost limitless.
Nanobodies, a tiny piece of the antibody cells found in the camelid animal family (camels, llamas, and so on), could interfere with just about any cellular organism, including bacteria and viruses.
One of those scientists reading the study results was Michelle Heck, PhD, Emerging Pests and Pathogens Research Molecular Biologist, USDA-ARS, who quickly reached out to her Florida colleagues, Robert Shattters, PhD, Research Molecular Biologist, USDA-ARS, and Marco Pitino, Lead Project Scientist at AgroSource. They were leading a team trying to find a way to battle citrus greening, an invasive disease with no known cure. The bacterial disease, vectored by the citrus psyllid, is slowly destroying the Florida citrus industry.
“The world was shut down, and we were texting with our group of scientists,” Heck says. “When we saw this paper, we were like, ‘Hey, what if we could produce these COVID-19 nanobodies in plants using our symbiont technology?”
A technology that holds promise to finally fight back citrus greening is revolutionary enough. A technology that can potentially treat any disease in any organism is a game changer.
Heck, Shatters, and Pitino set out to do the following: See if plants and symbionts could produce nanobodies against COVID-19 and citrus greening disease, and find a cost-effective way to deliver nanobodies to trees to fight against citrus greening.
The trio recently released their own study showing proof of concept for these ideas.
Naturally, we had a lot of questions on nanobodies. Here is a small part of the conversation we had with Heck and Shatters.
Q. What, exactly, are nanobodies?
Heck: All organisms have an immune response, an immune system. When most animals produce antibodies, the antibodies are gigantic. Camelids produce an antibody that has a very small domain with the function to recognize the antigen independently from the rest of the antibody molecule. And this is what's called the nanobody.
It's just this very tiny domain at the very top of the camelid antibody. Nanobodies are so small and structurally diverse. And because of their small size, they can do things in terms of protein interactions that larger antibodies can't successfully do. One of them is to move systemically throughout the plant’s vascular system, enter into cells, and bind to regions of proteins that larger antibodies can't have access to.
Shatters: They’re about 10 times smaller than a conventional antibody, to give you an idea of how much smaller they are, which is why they are so mobile. They can get into little pockets on the target molecule that an antibody can’t. And they are also much more stable.
Q. How do nanobodies work?
Heck: [In citrus greening], the bacteria express these little weapons proteins called effector molecules/ effector proteins. [An effector molecule is a small molecule that selectively binds to host molecules and regulates their biological activity and this activity can induce disease symptoms.]
If the bacterial infection starts here in the plant or on this leaf, the bacteria will secrete effector proteins that move to the other part of the plant to dampen the plant’s immune system and create an environment that supports pathogen growth.
And so, we hypothesized that if we developed a way to block those bacterial effector molecules from working, we could stop the infection process in its track.
We are very fortunate to collaborate with Marco [Pitino]. He did his PhD on effector molecules that aphids use to colonize plants. In fact, he was one of the first to discuss how these effector proteins work.
Shatters: The HLB bacteria gets in the plant. It produces these effector proteins that move out and induce changes that cause the disease. So, if you block those proteins from their function, symptoms don't develop, the bacteria can't replicate and so you've stopped the disease cycle.
“We're very frustrated. The citrus industry is going under, and the growers are suffering. I know personally some that went out of business.”
Q. A big part of your work was on how to deliver the nanobodies to the plant. What were you trying to learn?
Shatters: We realized that we couldn't just spray it, for a number of reasons. They're biological molecules. They're expensive. You can't spray and get, say, less than 1% taken up in the tree. It's just not feasible.
So, we stepped back.
[We considered] how long it will take to register, if will it be accepted, and so on.
We're very frustrated. The citrus industry is going under, and the growers are suffering. I know personally some that went out of business.
Q. So you needed something you that could make it to market relatively quickly and that was likely to make it through regulations. Your paper mentions using symbionts — living cells that need the plant to live but don’t harm it — as a delivery system. How does that work?
Shatters: One of the things that came up over and over was this idea of, what if we could make something like an insulin pump they strap on a tree and it delivers a proper dose over time? But we’ve got to do it biologically, because we can't afford to put a mechanical pump on every tree.
But if the tree could do that…
And so we came up with this concept of rethinking how people make transgenic plants. And that's what the symbiont is.
We take some cells from the plant, we modify them with genes to produce growth hormones, so they grow into what we call a symbiont. It's a group of citrus cells that grow where they are attached to the tree and do not move from that location.
So we engineer them to produce things that we want them to produce, molecules such as nanobodies or other molecules that can act as a defense against the bacterium that causes citrus greening disease. That’s our symbiont technology, and we’ve done it.
Q: OK, there’s something I don’t understand. You say this symbiont can be used on just about anything, including citrus trees and tomatoes. But how? A tree has bark, a cambium – its vascular system is significantly different from a tomato’s.
Heck: The application method we use involves sort of creating a small site of injury, a small bark flap, where we apply that group of cells. Since that group of cells is expressing plant growth regulators amazingly, it's enough to recruit the development of new vascular tissue right to that site of growing cells. They essentially adapt in their size and structure and texture to whatever host it attaches to.
Shatters: As Michelle said, it recruits the vascular tissue from the plant. It is important to know that this process is very natural and developed by mimicking something a bacterium has been doing to many plant types for probably millions of years. So this process has evolved to function on many plant types, even though, the structure, the morphology, of plants are totally different.
Q: And these cells can be created inexpensively?
Shatters: Wow! These cells are a tremendous bio factory. They produce large amounts of whatever we want them to produce. We could get them to produce things in in plants that may be very cost effective.
Our idea is that we could either culture these modified plant cells in fermentation systems for highly controlled production, or attach these to plants in greenhouse or field production for a much more cost-effective bio factory. It’s a highly scalable process.
We're now looking at pilot plant production of these in larger fermentation systems and working with engineers to help us understand it better. We developed a way to culture these where they can produce a lot of protein very cost effectively.
Q. Will the symbiont cells cause a threat to crops and the soil?
Shatters: Remove these cells from their host plant and they can't produce a plant. They can't produce roots. They can't produce leaves. They can't produce flowers, or pollen, or seed. They don't move, they don't migrate. If they fall off, they die.
Q. How far along is this research?
Shatters: We have trees in the greenhouse where we optimized the process and are moving to field production at our research farm this year. Greenhouse experiments demonstrate growth stimulation with lack of citrus greening disease symptoms. We have also been optimizing symbiont development on mature trees in the field at our research farm and have shown that there is no negative effect on citrus tree growth and yield in a two-year experiment.
We're now in the process of evaluating how to use symbionts to fight citrus greening disease. We plan on starting field trials this year using funding from a USDA, National Institute of Food and Agriculture grant.
Q: What’s next?
Shatters: We can produce these cells in the lab. And we can take some of these cells and apply them to trees. And they'll grow into a bump right there on the side of the tree where we attach them.
We're going into the field with a first generation of symbionts designed to fight citrus greening disease. We will be evaluating their performance over the next couple of years.
It’s important to know that our grant-supported work has an advisory panel made up of citrus industry representatives. We keep them informed about our advances and take advise on priorities and strategies to move this to commercial delivery as soon as possible. So we'll be tweaking this as we move forward. We're also in discussion with regulatory agencies. It's a new technology, but because we are not creating a whole genetically engineered plant and the engineered cells we do produce cannot escape, we believe that we will ultimately be able to significantly reduce the time it takes to get regulatory approval for symbionts.
We're hoping to get this out in a few years, so the growers have access to it.
