Hi everybody. And welcome to this week's talking biotech podcast by Calabra. Now about 9 years ago, we had on some of the first talking biotech episodes. And one of my favorite stories in biotechnology was the American chestnut. And the reason I like this story so much is because everybody had this idea that biotechnology was strictly the purview of companies that were going to make monstrous amounts of money off of selling seeds and and that this was the technology of Monsanto and and and you could really disqualify any argument by saying things like that or at least attempt to disqualify any good argument of about biotechnology by tainting it with some sort of weird financial interest.

But that's why I love the story of the American Chestnut because this was the vision of doctor Bill Powell and others who at the time were identifying that the American Chestnut had, was not on the radar anymore. We'll talk about that today. You know, what happened to it? But what was exciting was there was a biotech solution. And this was a biotech solution in conservation, which was super exciting because we were using it for something that everybody could agree on.

Right? Yeah. So, people still find a way to complain, but it still means it's a super cool project that's that is worthy of our attention and worthy of a good update. And so today, we're going to speak with 2 folks who are involved in the project. We're speaking with doctor Andy Newnhouse.

He's the director of the American Chestnut Research and Restoration Project, and Eric Carlson, who's a PhD candidate, who's been on with us before. He's, a PhD candidate in the same program at the State University of New York College of Environmental Science and Forestry. So, welcome to the podcast, Andy and Eric. Thank you.

Thanks for having me

back.

Yeah. You bet. Yeah. Glad you could do this. And I know it's, it's been a a challenging time in some ways.

We can get to that a little bit. And and, I really appreciate that you're able to join on with us, right now. So, anyway, just give me kind of the quick review of the backstory. Folks who are really interested could go look at the other, episodes in the series, but, you know, we talked about this before. And can you tell me, you know, where what was the American Chestnut in terms of its dominance in forests, and where did it go?

Yeah. American chestnuts used to be one of the most prominent, deciduous trees in eastern US forests, at least in some areas of the Eastern US. They were really unique in their size. They're some of the biggest trees in, in the Eastern US at least. And also really unique in terms of making a food crop that was actually attractive to people and a variety of wildlife.

So really filled an interesting ecological niche. And as far as where they went, people don't see them much anymore because they are mostly wiped out by an invasive fungal pathogen that, was introduced to the US in the late 1800 and really started moving through chestnut populations in the early 9 early first half of the 1900.

Yeah. I used to be that, and I always heard this said that a squirrel who was ambitious could travel from Georgia to Maine strictly on the treetops of the American Chestnut, and now they have to hitchhike. There was that dominant.

Verify that, but, that's, right. The the regardless, that illustrates that people were really recognizing the importance of these trees. They've really, taken an important part in our, culture and history.

And are these trees extinct, or are they still out there somewhere?

There's still many trees out in the forest, but they're mainly reduced to stump sprouts. So the blight will kill the tree above the ground, but it's unable to kill the roots because of the microorganisms in the soil prevent the fungus from killing the roots and chestnuts have the ability to stump sprout. So these large trees were killed to the ground and now you can still find many of them in the forest repeatedly stump sprouting as kind of like a small bush. And some of them, if they find sunlight, they can actually get big enough to flower, but, inevitably, they've gone reinfected and killed back down to the ground in a repeating cycle.

Yeah. And and so that you still have trees that are there, but just the genetics are alive somewhere just, really reduced in vigor. But you have, vigorous species of chestnuts that are interferral, things like Chinese chestnut, which are resistant to this fungus. So why not just breed a better chestnut by using, the Chinese, resistance genes and breed those into American Chestnut.

Yeah. That's definitely been attempted. And I would start by saying that Chinese chestnuts are considered resistant to blight, but they're not fully resistant. Chinese chestnuts and every species of chestnut can die from blight depending on the severity of the infection and environmental factors and so on. So you're not starting with perfect resistance, I guess, for a breeding program that's been, attempted repeatedly in different ways over many decades.

And a couple of the biggest challenges with that are that there are several different genes in Chinese Chestnut that all work together sort of or all interacting to provide light resistance in that species. So it's not a matter of just a couple of genes getting into off breeding program. There's so many genes located in so many different parts of the genome that a, that breeding for bite resistance is not a trivial exercise. Even if you could find an offspring that had the perfect combination of traits of of traits that people like about American Chestnut for restoration and suitability for us, for wildlife and so on and the blight resistance of the Chinese Chestnut, that could exist in one tree. But as soon as that starts crossing with wild relatives that we hope would be a part of an ongoing restoration effort or reintroduction effort, then the resistance from that Chinese ancestor would would be, reduced in each subsequent generation.

So it's it's really gonna be a difficult thing to use breeding alone to address flight resistance.

Yeah. I I asked that kind of as a devil's advocate kind of question because you can hear the opponents of biotechnology saying, well, this is a breeding question. The other big one for me is how long does it take from seed through juvenility in a chestnut before you can be, sexually competent to be able to produce pollen to even begin the breeding process?

Well, normally, in field conditions, it can take up to 8 to 10 years before trees begin producing female flowers so you can produce offspring from them. So it's a quite a delayed process and there's a long wait between generations.

That said, we're working on speeding that up. We're able to produce pollen indoors in sometimes less than a year. And, so we're that's getting faster. It's not always 8 to 10 years for a generation, but it's still certainly slow compared to agricultural crops or other species that might be more familiar.

Yeah. It makes for a long PhD project. You can retire when you finish turn in your dissertation. I guess the the other big question is where did the idea of using a biotechnology approach come from? I I don't know the exact genesis of this, but I know it's been going on for a long

time. Yeah. The original idea probably came from a couple of chestnut enthusiasts, not academics, not scientists, people who got excited about chestnut trees by finding them while they were out hunting in Western New York. It's my understanding of the story. This is the late 19 eighties, and these enthusiasts approached a couple of different scientists with with the idea of using these, at the time, brand new techniques of modern biotechnology, as opposed to, traditional breeding that was started at that time, but in very early phases of that, that project too.

And so these these, enthusiastic, naturalists, I guess, ended up talking to our predecessors here at SUNY ESF, Bill Powell and Chuck Mater, and, introduced this idea of using biotechnology. And it's been here and we've been working on it ever since. And the the, I guess, breakthrough, time that that became realistic was when, Bill Powell specifically realized that a mechanism called oxalate oxidase that uses a gene from wheat, but is widespread in a variety of other species as well. That this gene could be effective in, reducing the symptoms of chestnut blight. That it can increase tolerance in American chestnuts enough with a single gene to, to potentially be a viable restoration effort.

Yeah.

We maybe we should note that, doctor Powell recently passed away, and and it's really, a tribute to him to see that project going forward. So it it really means a lot to to, you know, me. I'm sure others in the field to to see this work continue because, you know, he was such an advocate to seeing it to fruition. And and, what exactly is the appetite for, say, the institute or other places to really keep carrying on this work?

We've seen a lot of support from our institution at every level. It's really an honor for me to, to direct the project and to carry on, the legacy that Bill started. He was a huge proponent for not being an individual face of the project. It wasn't a one person. It wasn't his project.

It was the team effort. He would always do a good job of recognizing the the team teamwork. So we really wanna to carry that on. We have a strong team. So the the researchers that are already working on the project, really excited to carry it on.

And then, the our university as an institution is really proud of the work and very supportive as well.

And outside of the institution, we've gotten a lot of support from people who follow the project and and, want doctor Powell's work to continue. So we're we're gonna keep pushing forward.

Yeah. And and I think doctor Powell, he was maybe in episode 10 of this series. So we're in the mid 400 now, and he was one of the first people I wanted to have on because of, his vision and and the excitement about the project. So with that in mind, let's take a break here. So we're speaking with doctor Andy Newhouse and Eric Eric Carlson from the State University of New York College of Environmental Science and Forestry.

This is the Talking Biotech Podcast by Calabra, and we'll be back in just a moment. And now we're back on the Talking Biotech Podcast by Calabrio, and we're speaking with doctor Andy Newhouse and Eric Carlson, the future doctor Eric Eric Carlson of the State University of of New York College of Environmental Science and Forestry about the blight resistant American Chestnut and the biotech tweak that may allow this tree to one day repatriate at least a part of Appalachia and restore some of the native ecology that really made residents of this particular tree species. So very exciting work. And we left this really talking about the genesis of the project and the problem that there was to solve. What exactly is the biotech tweak?

And you mentioned oxalate oxidase. What exactly is that tweak? Where did the gene come from, and how does it work?

So the gene that we're using is called oxalate oxidase, and it's used to counter the main virulence factor of the blight fungus, which is oxalic acid. When the blight infects wounded tissue, it begins secreting this strong organic acid called oxalic acid, which, has several effects on the tree. It kills the tissue and it chelates the calcium out of cell walls and essentially just weakens the tissue. And what oxalate oxidase does is it degrades that acid into hydrogen peroxide and c02. And there's 2 important things there.

One is it's neutralizing the acid, which is causing the damage, but it's also releasing that hydrogen peroxide. And hydrogen peroxide is a defense signaling molecule, and it kind of tells the tree that this infection is happening and it will activate a suite of defense genes that are already within the chestnut to begin compartmentalizing that blight and protecting the tree.

Yeah. And here's a really cool part that I like about this is that sometimes opponents will say, well, you're targeting a fungus. And if you, remove this fungus, then you're throwing off the ecology of all the other things this fungus may do in terms of, its role in the ecosystem. But all you're really doing is disarming it. Right?

You're you're just keeping it from harming the tree. It's still there doing its thing and participating in all of its other fungal business. But so so how important was that in the design of this approach?

It was it's definitely been important kind of in hindsight. At the time we were first starting to work on it, we were looking at various approaches toward enhancing flight tolerance, and it happened that this one worked. But I think it's really significant as a first step for using biotechnology for something like this that, like you said, it's not even harming the blight fungus. And in this case, the fungus is non native, and so people probably wouldn't be arguing for its importance in native ecosystems. But, a very valid, argument is that we don't want to be interrupting relationships or, functions of other fungi in the ecosystem, especially things like mycorrhizal fungi that associate with tree roots, but other fungi in general.

And so in this case, if the mechanism is specific to the pathogen and doesn't even harm the pathogen. And we've done a series of tests looking at mycorrhizae that, that confirm this, that the transgenic trees are forming normal mycorrhizal relationships, that the Oxidoxidase is not interrupting or preventing these associations. And that's, that's been consistent and an important part of of thinking about using this for conservation.

No. Very cool. And I guess the other other really good use of this particular mechanism is in the question that always comes up is, well, what if the fungus becomes resistant to the to the, mechanism? And so what are the odds of that happening relative to say other types of either chemical control or other type of genetic engineering approaches?

Well, compared to other approaches that might use some kind of, like, fungicidal activity, the selection pressure is much less for the fungus to overcome the resistance since you're not actively killing the fungus and selecting for cells that have resistance to the mechanism. You're allowing the fungus to continue to live and reproduce on these trees in a way that, doesn't select for individuals that are resistant to the mechanism.

We still see cankers on transgenic trees. They're not we we don't use the term resistance usually because they're not resisting the fungus. They're not preventing infections. We see infections, but they look different. You can see that the tree is responding, it's swelling, it's, it's using some existing mechanisms and the oxalate oxidase to, to tolerate the blight infection.

In some cases, these transgenic trees will get severe cankers and die from blight. But on average, we see these more superficial cankers that aren't causing damage, and the trees look, better than anything else we have so far.

Yeah. That's a that's a a really good point. So resistance is where you don't get the infection. Tolerances, you get the infection, but you don't care. And I think that's a really important point, in this discussion.

And and when you mentioned that you see some trees that do succumb, is that because there's some genetic heterogeneity within the population that is, that is maybe harboring the transgene, but still, still succumbing? Or is it something that maybe has to do with the environment that a tree is in, or is it both?

It's probably both. There's a classic, example, I guess, used in, plant disease classes of what's called the disease triangle, where there are 3 factors that, that affect the outcome of a disease. And there's the host or the tree in this case, and that's gonna vary in terms of how much kind of background resistance it has. Maybe there are family effects there, and then there's the pathogen itself. There are different strains of the blight fungus.

Some are more severe than others, and then there's the environment as the third point in that triangle. And any of those things can vary and affect the outcome of the disease, whether the tree survives or doesn't.

Yeah. And you can't control the environment and you can't control the pathogen necessarily or you choose not to in this case. So you you bolster the genetics, but it isn't perfect and then, you know, it does what it does. It's good. So can you tell me where we are in terms of the release process?

Because I remember this was just kind of getting underway last time we spoke. So where are we now?

Sure. We are waiting on regulatory review by 3 different federal agencies at this point. The USDA, the EPA, and the FDA all have to review this tree before it can be widely distributed or or used for restoration purposes. We are able to do testing outside under permits, but that's pretty limited. The the confined field trials are an important step, but not really a meaningful part of restoration.

So, we're waiting on this review, and, it's been ongoing for just about 4 years now. And and there's not a specific time frame that's provided, but we, expect a decision hopefully pretty soon.

And and the FDA, that seems kinda weird. Is that because people eat chestnuts?

Exactly. Yeah. FDA wouldn't be involved in other types of trees, except that this is a food product for people, and in some cases, could be used for animal feed as well. So they're reviewing the nutritional safety of chestnuts.

So you you had this long 4 year path and, you know, the regulatory hurdles are things we know about. But have there been any other setbacks in this process that really kind of, slowed it to get to the point where it is?

Labeling? Yeah. The there's been a recent finding. We've recently determined that the trees that we've been working with, or at least most of the trees we were working with, were labeled incorrect. When we first developed these trees in 20 15 or so, we were first doing pollinations in 2016 to get to the offspring generations that we've been testing.

There were 2 different lines that we developed at the same time. They were called Darling 54 and Darling 58. And they came from the same experiment. The the genetic backgrounds are the same. The new insertions of DNA are the same.

The difference is where the new DNA, where the transgene got inserted. And so it's located on a different chromosome. We recently learned that what we've been calling darling 58, that most of those offspring trees are actually darling 54. So we are working to correct that. We have submitted, new information to the USDA so that the application the the petition we've submitted is correct, and that's certainly been a challenge.

But the important parts are that the trees we've already tested are the ones that we want to, submit. So we've done years of work on Darling 54. That's the correct name. We had it was incorrectly labeled at the time. But all of the results, all of these mycorrhizal tests that I mentioned, the, nutritional safety, the various, environmental interactions that we've looked at have been tested on DARLING 54.

So we're confident about the safety and the effectiveness of Darling 54, and we're, working to correct the the name. That's been a challenge. It's been a somewhat of a delay for the regulatory process. We don't know how much difference that'll make, but we have been in close touch with the regulators and are working to resolve that.

Yeah. And and and I can imagine how this happens. You know, you're growing tons of trees. You've got multiple hands and, working on these things, you know, multiple people of different, and it's it's so easy to do. But nonetheless, glad you got it sorted out.

But where's what's the future of the project at this point? Where are we now, and where do you expect to go next if you get approval?

Yeah. We are excited to continue improving the existing chestnut lines we have. So Darling 54, has room for improvement through traditional breeding, through selecting some of the best individuals from the population we have for subsequent generations, And I hope Eric can talk about some of his work with kinda next steps of Chestnut. And then we're also looking at expanding to using biotech approaches for potentially other species, for, other primarily native northeastern trees that are under threat from, pests and diseases?

We have our Darling lines which have the oxalate oxidase gene, regulated by constituent promoter. And that promoter gives us very strong expression throughout the plant at all times and it gives us very good resistance. But, shortly after we developed those lines, we also developed a new gene construct that uses the same gene, but it's using an inducible promoter from poplar. And so that promoter keeps the gene turned off at most times. But when the trees become wounded or infected, that gene gets turned on and turned on at a really high level.

And we see resistance or tolerance levels that are similar to our darling trees. So that's kind of our next tree that we're working on. We we kind of have a fun name for it. We named it, Darwin. So the DAR is obviously an homage to our darling trees, and the WIN is for the WIN promoter, which is the name of that genetic element that we took from poplar.

So that was actually a name that doctor Paula came up with.

Yeah. Kinda clever. That's good. And, you know, and we mentioned in the beginning that there's a little pushback against this technology, which just seems so strange because it seems like such a valiant effort to wanna repatriate a forest with the trees that are supposed to be there that we wrecked. You know?

Humans brought in this fungus and and and, you know, and and so it's our fault to break it, and now we can fix it. What are the arguments that are used to push back against this concept?

Yeah. The arguments are more philosophical than scientific, I think. And so that's a really, interesting part of the project for me to deal with some of the social considerations because that that really matters. Even if we have the best science and we can show that the trees are safe and we can show they're effective, If people aren't interested in using them for restoration, then the project isn't really going to be successful. And so, in some cases, we can do tests, we can do experiments, and show results, and address some of those concerns.

But in other cases, the concerns are more about our place in nature, whether, like, the the the rationale you described, this was a mistake caused by people, and we can do something about it. I agree. For me, that's a lot of what keeps me going and what's so important about this project. And and becoming increasingly an environmental, concern that we need to start dealing with some of these problems that are that are only increasing as time goes on. But for other people, the the concern might be about what roles we should be playing in nature, and about how much we should be intervening.

And that's that's a harder thing to address with science. It's important to talk about, but also important to keep in context of alternatives. So if people are agreeing that we should do something about chestnut blight or about something or do something about chestnut restoration, then what options are there? There's traditional breeding, which involves, crossing with a non native tree that is probably safe and effective, but has the has some other, concerns as well. And and really a lot of the, potential scientific concerns that people have about genetic engineering might be equally applied to traditional breeding or or other approaches that that get discussed.

Another common thing you hear is the concern for unintended consequences, that you can't predict every single consequence that will happen as a result of restoring these trees. And it's it's obviously impossible to know every single consequence that's gonna happen, but you can rule out some of the worst stuff by doing safety testing. Like, we we we tested these trees, with amphibians and insects and and and pollinators. And we were able to see that there was no unexpected unexpected toxicities or anything of that nature. And so we can rule out the worst things and and but you can't necessarily know every single thing that's gonna happen, but we know that at least they're safe.

Another, kinda response to the unintended consequences argument is that we do have a pretty good idea of what will happen if we don't do anything. If we continue this path of of kind of ignoring the problem, it's not going to solve itself. Chestnuts are not going to, spontaneously generate blight resistance, at least not American chestnuts. So, the if if your options are to not do anything or to use a modern approach and do something, well, there would be consequences to both of those decisions. There isn't a free pass of something that's guaranteed to be safe.

Yeah. I I love that answer because this is not the last problem humans will create. And it could be used as a model for the way in which we navigate a situation to restore ecology or maybe bring back something that we've lost. And I I think it's just such a important opportunity for us to sort through the philosophical challenges, the scientific challenges, the way in which we address it from a sociological and psychological side, the way in which we communicate it properly. All these things are so important, and that's just what I love about this project in a nutshell.

Right? So if people wanna learn more about it, where can you direct them to online resources?

Our, our website for the lab is atesf.edu/chestnut. We have, many pieces of information there and links to different things. We have our progress reports and our, scientific data releases there. We're also on, social media. On Facebook, we have the American Chestnut Research and Restoration Project page and group, which you can check out where there's lots of updates shared there.

And, also, there's a community of people that like to discuss chestnuts and and all the topics surrounding that. So those are a couple of places where we can find our stuff. Also on Instagram.

Very good. So I would encourage listeners to please, please, please, follow those pages because the more numbers of people who follow and share this stuff, the better it gets for everybody in terms of ushering through this technology when it becomes available. And it'll help all of us once it gets approved. It'll help it find the field even faster, which is really pretty exciting. So, doctor Andy Newhouse and Eric Carlson, thank you so much for joining me on the Talking Biotech podcast.

I love the story. I'm so glad you're pursuing it, and keep us posted of any future developments.

Thanks. Thank you.

And for everybody listening, thank you for following us on the Talking Biotech podcast in our new format with video and audio. And this should be if you're listening into the normal format, go check it out over on the YouTubes. We'll be on over there as well. But most of all, follow the project. Follow the, American Chestnut Project.

It is a model for the way in which we may solve future problems. It's also a tribute to doctor Powell who, had such a vision and was so excited about seeing this work go to fruition. So, thank you very much for listening to the Talking Biotech podcast, and we'll talk to you again next week.