Subscribe
Share
Share
Embed
Talking Biotech is a weekly podcast that uncovers the stories, ideas and research of people at the frontier of biology and engineering.
Each episode explores how science and technology will transform agriculture, protect the environment, and feed 10 billion people by 2050.
Interviews are led by Dr. Kevin Folta, a professor of molecular biology and genomics.
Kevin Folta (00:02.797)
Hi everybody and welcome to this week's talking biotech podcast. And we look back into the seminal discoveries in biotechnology. There were some things happening in plants and around plant pathology long before many of the newest techniques were even on the radar. This was happening in response to an infection that was happening normally in plants that seems to exploit certain weather events to part to take advantage of.
part of its infectious cycle. And today we're going to be talking with, let me try that again. I'm kind of stumbling over my words. I'm good with this normal. Here we go. Hi everybody and welcome to this week's Talking Biotech podcast.
Long ago, there were some interesting developments in plant biotechnology or actually more microbial biotechnology as they related to plants and plant pathology. And even though over the series of this podcast, we've discussed many innovations about plant protection, whether it was from the papayas and protecting them from ringspot virus to other innovations around Higgs or other types of protection for plants. Some of the innovations were pretty early.
even back in the 1980s. And today we're going to talk about that. We're speaking with Dr. Steven Lindo. He's a professor emeritus from University of California, Berkeley. So welcome to the podcast.
Steven Earl Lindow (01:32.216)
Hello.
Kevin Folta (01:34.231)
Yeah, so thank you very much for joining me. This is exciting because every semester and every year in my course and my plant fizz class, we talk about ice minus and ice minus bacteria and talk about the role of ice formation in plants and how that works and how plants deal with it. And let's start out there. Let's talk about freezing and how it damages plant cells. Can we start with this idea of nucleation and why that's damaging to plants as we get around?
below freezing temperatures.
Steven Earl Lindow (02:05.678)
Yeah, it simplistically, I always look at plants and breeding in two groups. Plants that can tolerate ice formation and plants that cannot.
Many plants like the grass in your lawn that can freeze during the night, you'll look frosty. It tolerates ice and ice forms only in between the plant cells. Whereas many of our more important crop plants cannot tolerate ice and their ice spreads unrestricted in the plant and eventually can enter the plant cells and cause them to be physically disrupted. basically frost damage is really very different depending on what types of plants we're talking about.
Kevin Folta (02:44.724)
And the idea of nucleation. how does that play a role in this? That water is surprisingly fluid, even below freezing, but nucleation seems to be the problem. So can you explain what that is?
Steven Earl Lindow (02:59.724)
Yes, ice is essential to have frost damage in any type of plant. And we were all tied in correctly that water freezes at zero degrees Celsius or 32 Fahrenheit. But that's not true. Water can super cool. It remains in a liquid state to quite cold temperatures. And small amounts of pure water can cool to around minus 40 degrees Celsius, which is quite cold. And certainly the water within plants we now know can cool to at least minus six or minus seven degrees, quite
routinely before ice is going to form. And to get ice to form in water that will otherwise super cool and remain liquid, we need a catalyst or an entity upon which water molecules can be trapped and hold into a ice-like structure. And then if the water is viciously cold, that water will then eventually freeze and spread into that entire volume of
Kevin Folta (03:57.646)
Yeah, so it's about something that is present that kind of can allow water to begin to structure into a crystal, Like orient with itself. And what is this normally in a plant?
Steven Earl Lindow (04:10.626)
Well, in till of our work, we didn't really know what cause I used to form.
It's long been known that certain minerals can cause ice nuclei or ice embryos to form, but they're not active at very warm temperatures. They cannot catalyze ice until it gets to around minus 10 degrees Celsius or colder. But we know that ice forms in plants at much warmer temperatures, and it was really only our own work that showed that it really was certain species of bacteria that act as a very, very efficient ice embryo or ice nucleating agent.
Kevin Folta (04:44.825)
Yeah, and that's where I wanted to go next. this bacteria, Pseudomonas, it's notorious for being able to cause damage in the temperature range we're talking about. So you get down right below freezing. then this bacteria has a protein which can actually spur that ice nucleation event. And so what was it that led you to look at that? I mean, to look at Pseudomonas first.
rather than some sort of just atmospheric anomaly that would lead to lead to the ice formation.
Steven Earl Lindow (05:20.014)
It's rather a long story about how we discovered the role of ice nucleation bacteria in freezing.
started in the early 60s in Wisconsin, University of Wisconsin, before I started graduate school. Inoculant of a fungal pathogen was being applied to young plants in the field to start epidemics of this fungal disease. And they had inoculated the dried powdered corn leaves from the previous season onto these plants. And it was kind of a freaky spring. It got cold. And only those plants that they had dusted with this powder froze. And the ones that hadn't
and dusted didn't freeze. And that was really the origin of my own thesis research was figure out what was in these powdered leaves that made other corn plants freeze. And kind of a long story, but it turned out that we could isolate this bacterium from the powdered corn leaves. The bacterium was surviving in these dried powders. And we inoculated eventually with a particular strain of bacteria that we found that I keyed out them to be Pseudomonas syringae that turned out to be very,
efficient at catalyzing ice formation and causing those plants to freeze. So it was really the lack of bacteria that was the exceptional situation. The inoculation process had provided the bacteria to catalyze the ice and we eventually found out what was in that inoculum and it was these bacteria.
Kevin Folta (06:47.641)
That's really cool. which department were you in at Wisconsin?
Steven Earl Lindow (06:51.286)
I'm trained as a plant pathologist and I was in the department of plant pathology at University of Wisconsin.
Kevin Folta (06:56.929)
Yeah, I banged around there a little bit too. I was up in Burge Hall up on the hill there, so.
Steven Earl Lindow (07:00.942)
It's a great pleasure to go to graduate school.
Kevin Folta (07:03.053)
Yeah, I was a post-doc there and I absolutely love that school. It's a great town too. So when you're talking about bacteria, how does a single protein do this? Or is it a bacteria, so maybe I rephrased the question, is it the bacteria itself or is it some sort of protein entity on the bacterial exterior?
Steven Earl Lindow (07:08.64)
Everybody liked medicine as well, yeah.
Kevin Folta (07:27.361)
that really is catalyzing the formation of the ice nucleus at temperatures where water would normally be liquid.
Steven Earl Lindow (07:34.455)
Yeah, it's our own work on the mechanism of isonucleation. We found that a single gene was required to cause this process. That single gene encodes a fairly large protein, and our biochemical work showed that it was located in the outermost part of the bacteria. So it's facing the outside world where it would interact with water outside of the bacterium. But it's a single, very large protein that has the ability to bind water and hold
it into a structure that looks like other little ice crystals. So binding water basically makes an ice embryo that, if sufficiently cold, will then grow into the bulk water.
Kevin Folta (08:17.977)
See, that's really cool. So what was it that led to the biotech solution? I mean, to modify the bacterium to not have this. Can you explain that whole process?
Steven Earl Lindow (08:31.534)
Well, we got into the biotech area basically just to test an ecological question because we'd already found that knowing that ice nucleating bacteria were the triggers for frost damage on plants like corn and flowers and so on, we could compete against them using other non-ice nucleating bacteria. Most bacteria don't catalyze ice, but their presence on a plant where the bacteria normally live compete and prevent the growth of the ice forming bacteria.
So with that in mind, we are very interested in finding the best possible competitor to keep the isonucleating bacteria from growing. And our thinking then would be that start with an isonucleating bacterium which knows exactly what it wants to do to live and grow on a plant, remove its deleterious trait, in this case, a single gene for an isonucleating protein, and apply it to a plant before they become colonized. And we could then have a preemptive competitive
exclusion. We're going to basically have a prophylactic shield to prevent the growth of other bacteria on those same plants.
Kevin Folta (09:39.361)
And this was back in the 80s, I mean, so what kind of techniques? And this is a geeky audience that enjoys hearing about molecular biology. how was this done back then? I mean, we weren't doing things like CRISPR, right? So how do we do these things back
Steven Earl Lindow (09:54.861)
This was very, very early in the days of recombinant DNA. I was very fortunate. So my colleagues, including my close colleague, Brian Staskowitz, had access to some of the very first cloning vectors. This was before genes were really routinely cloned. Some of the very first genes cloned were our isonucleating bacterial genes. So we were able to clone and characterize the gene. But using the gene as a mutagen, we could then use it to make a very precise
modification of the genome of the bacterium. So we take the gene, it out, chop a chunk of the gene out of the middle and then reintroduce that back into the bacterium where a recombination process would allow a precise replacement of the functional gene with our deletion containing gene. So it involves recombinant techniques, but you end up with a mutation which could have occurred naturally.
Kevin Folta (10:44.718)
Yes, so.
Kevin Folta (10:51.915)
And this is what's so interesting. mean, this is even predating PCR, right? I mean, this is
Steven Earl Lindow (10:56.482)
This was before PCR, yes?
Kevin Folta (10:58.519)
Yeah, so this was a good old-fashioned molecular biology. So taking a chunk of this thing, creating the deletion in the lab, and then using homologous recombination to put it back in. Yeah, so that, wow, that's really cool. So I can still remember those days. We did our first PCR in two water baths using a clean-out fragment. So you probably did the same. Yeah, that, so.
Steven Earl Lindow (11:08.588)
Exactly.
Steven Earl Lindow (11:17.454)
expensive machine.
Kevin Folta (11:24.011)
If let's let's take a break there. And when we come back on the other side of the break, we'll talk about the greenhouse trials and how this eventually made its way or maybe didn't make its way into the field. This is the Talking Biotech podcast. Be back in just a moment. That's very cool. This is great. I'm glad we got to talk about this. And then on the other side, we'll talk about the trials and all that good stuff. Here we go.
And now we're back on the Talking Biotech podcast. We're speaking with Dr. Steve Lindow. He's an emeritus professor from UC Berkeley, and we're talking about ice minus bacteria and how these bacteria were used.
discovered to suppress ice formation on plants and how they may be used in ways to protect plants in the field. And that really leads to where we go next. So how did the early trials of this work? You alluded to this a little bit, but when you had these ice minus bacteria that were now competing for ecological space on a given crop, how did it work?
Steven Earl Lindow (12:29.064)
Well, we had done extensive greenhouse study to show that in fact we compete against isonucleating bacteria. And again, our expectation was that those bacteria that were most similar to the isonucleating bacteria would be the most easily prevented from growing on plants.
But what was not clear was how diverse the isonucleating bacteria were on plants in the field. How could we try to shield them against all possible isonucleating bacteria? Pseudomonas syringes is most common isonucleating bacteria, but there are other species of bacteria that also have the gene and can cause freezing. So our real interest then was to take us eventually to the field after our greenhouse work to test whether we could equally well
compete against all the ice nucleating bacteria that a plant might encounter in the field.
Kevin Folta (13:24.033)
And that's a really good point because what other types of isonucleating bacteria are there and are there are there are they as common as Pseudomonas syringae, at least on crop plants?
Steven Earl Lindow (13:35.151)
Pseudomonas syringae is the most common and it's also the most active. But depends if you're a lumper or a splitter in terms of naming bacteria, there's three or four other bacterial species related pseudomonas species and then a couple of somewhat unrelated species including Xanthomonas compestris translucent, a barley pathogen, and also a non-pathogen.
which is now called Pantoia aglomens and various other Pantoia species can also nucleotide that can also be found on plants.
Kevin Folta (14:09.497)
So those would be potentially other ways in which this nucleation event could occur but could be suppressed by a larger population of Pseudomonas so that at least that one's not playing in the game, right? So, okay, so you make this cool recombinant bacterium that's got a new way of suppressing this isonucleating protein and in 1987 the first ice minus trials were
Steven Earl Lindow (14:20.865)
Exactly.
Kevin Folta (14:33.635)
performed and they were met with a lot of protest and there were field trashings, legal challenges, folks like Jeremy Rifkin were involved. And looking back on this, do you think the scientific community could have had a little bit of different messaging and the safety and maybe done the trial differently or was this just an inevitable controversy in the pre internet days? Tell us a little bit about the discussion that was happening in the public and maybe just around the university about this.
Steven Earl Lindow (15:03.502)
Well, this was very much of a landmark. It was the first recombinant microorganism to be released into the field. And essentially, was very much precedent setting. And it got a lot of attention. Before this podcast, I probably have done 200 300 television interviews over the years at the time to help educate people. There was just an awful lot of attention. And this was pre-internet. I can't imagine what it would have been like.
anybody can do anything on the internet. But yes, there was an awful lot of attention from all around the world. But it was also a really first glimpse at what we see so much of today, which was kind of skepticism or distrust of experts.
In other words, to get our experiment eventually approved for use in the field, we had to go through a very elaborate process of review by a number of different agencies. And there were experts of all types that looked at this. And we had done lots of educational outreach experiments to try to tell people what it was about and try to convince them that, like the other experts, that there should not be too much to worry about. But despite that, there will always be people that are worried or distrustful
experts and that's only grown since.
Kevin Folta (16:22.499)
Yeah, I could not imagine trying to get this released, you know, in the current environment, especially with many layers of distrust of expertise and trashing of our institutions. I'm up against it all the time, you know, just trying to talk about science to make people feel a little better about their food and about farming. But let's go back to your work. This was the.
first ever authorized release of a genetically modified organism and the moment you were finally allowed to spray this on strawberries and actually do this work, did it feel like a scientific triumph or was it just like I'm finally done with the legalities of getting that done?
Steven Earl Lindow (17:07.874)
This was very much a slog. It was about six years of dealing with different regulatory agencies, getting experimental use permits. I was very fortunate that the University of California helped, at least in some of the more legal aspects, because there was this one court a number of different times to test some of the environmental regulations that were being applied for the first time in our study. But when we were finally able to do our city, was done on potatoes.
Northern California near the Oregon California border. It was a bitter relief. Like I could say, it was a real slog. It had taken years out of my professional career. And yes, I got a lot of attention for it, but it distracted me from doing the other things that I have to do, including teaching and other research.
Kevin Folta (17:59.118)
Yeah, I can imagine. how has all the controversy really contributed to, like say, the coordinated framework of regulation and biotechnology that's still in use today? And do think that those early hurdles and the early perceptions and the early discussions really made today's regulations better or maybe unnecessarily more restrictive?
Steven Earl Lindow (18:26.158)
Well, I looked positively on that and that I was going through these many hurdles. And it was at the time when you had the different regulatory agencies and governmental organizations were parsing out what aspect of biotechnology and all who would regulate. So that's why I had to first deal with the National Institutes of Health and eventually with EPA and USDA. And that has now been.
sort it out that there is a partitioning of attention on different types of experiments by different agencies or institutions as appropriate. So that was good, but it was a very painful process when that early, what I would consider seriously over-regulated regulation were now been parsed out to at least there's a little, there are fewer eyes on any individual type of an experiment.
Kevin Folta (19:22.041)
Well, the commercial product that emerged from all this was called Frostban and it was never really marketed even though it worked. Right. I mean, is that where and what does that story tell us about the gap between successful laboratory discovery and viable commercial application of genetically engineered microbes?
Steven Earl Lindow (19:41.901)
I think that's still a remnant of the overregulation that remains in place.
There are a number of biological pesticides and our recombinant ice minus bacteria is considered a pesticide by the US EPA. So to be mitigating a pest, ice nucleating bacteria are considered a pest because they can cause frost damage. So our competitive bacterium is considered a pesticide.
And as such, to be used on commercial agriculture, it has to receive a license from the US EPA under FIFRA, the Federal Insecticide Redenticide and whatever act. Now unfortunately, there are relatively straightforward processes to get a permit eventually for a chemical pesticide or even naturally occurring bacteria.
But the rules are still yet to be fully fleshed out for recombinants. And so it's kind of an emerging process. And so there was so many uncertainties of what would be needed to fulfill the requirements of EPA that to this date, there have been very few of any recombinant microbes of any sort that's ever been used because of the uncertainty of the requirements. And so.
Is it going to be $1 million or is it going to be $50 million worth of these types of experiments that would be required? And because it would require a commercial sponsor to produce and actually pay for the kind of toxicology and other environmental tests to show their efficacy as well as safety, most companies that might have been interested in further developing this said, come back when we know what we're up to.
Kevin Folta (21:37.626)
It's really interesting that these were the questions that were being asked back then and still we don't have an easy way to solve those questions because the next thing I was going to ask you about was are there any contemporary efforts to revive this strategy? It still seems like something we could use. believe down here in Florida
We have freeze events and frost events that are just teetering around that, you know, zero Celsius level where farmers have to do a lot of protection and they lose a lot of crops on things like strawberries that are produced in the winter. And is something like this still commercially possible?
Steven Earl Lindow (22:18.698)
It really kind of depends on what you call recombinant. And that the world is still kind of feeling their way around. And there's recently been a bit of relief on regulation of things that have been modified by, CRISPR technology. It isn't necessarily considered recombinant. And it is something that could have been formed and produced by a natural process, just like ours was a natural, undistinguishable from a naturally occurring deletion mutant.
a bit of a re-examination of what is needed to be classified as a recombinant organism. But there's still a lot of sensitivity to using anything that would have come from the lab in the field, and that still kind of hurts the field.
Kevin Folta (23:08.025)
Well, you were one of the pioneers who probably took a lot of heat from anti-biotech, anti-GMO folks. I don't think the folks who did insulin got the same kind of pushback.
And so what advice would you have or what lessons did you learn in the 1980s that today scientists should really think about because we still have this huge pipeline of outstanding innovations in plants and microbes that just aren't getting to application. And so what do we know from 40 some years ago that could really be applied today to maybe accelerate this process?
Steven Earl Lindow (23:48.943)
Well, I think we still need to somehow cultivate a better recognition of what the scientific process is about and how scientists are constantly policing themselves. And there are a lot of regulations and smart people looking at all of these types of procedures and products to look at their safety. We did a lot of outreach. I did way more than my share of meetings with local groups and so on.
to try to explain the science, but not everybody can be an expert scientist. For example, I know very little about radiation safety, but I do know that there are a lot of radiation safety people out there that I am comfortable with keeping an eye on those kinds of risks for me. I think people just have to become aware that there are a lot of experts out there that should be trusted and by...
Having the right people look at these that should be looking at these things, think we can all feel quite safe about moving forward with any type of technology.
Kevin Folta (24:58.553)
Was there any other thought, anything else that I should ask you about that maybe I didn't touch on? Anything else, any other questions I should ask you? Any other kind of, I don't know, is there anything, anything I missed?
Steven Earl Lindow (25:13.91)
No, I don't think so. It's pretty much covered to main issues.
Kevin Folta (25:15.491)
it's
We talked a lot about mechanism and things like that and all that's good. But I think that's a good place to wrap up. So let me just thank you here and then we'll go from there. Okay, here we go.
Kevin Folta (25:34.041)
Well, it's kind of a, let me go again. It's kind of a shame that I've gone 500 episodes almost into this without talking about the ice minus bacterium. It's always been a story. teach it in my classes and it's always been something out there and I'm really glad to be able to talk to you about it and kind of meet you here to talk about. So Dr. Steven Lindau, thank you very, very much for joining me today and best wishes going forward.
Steven Earl Lindow (26:02.382)
Good luck on your projects.
Kevin Folta (26:04.533)
And to everyone listening, this is one of the early ones. And so get familiar with this story because this is a great example of how biotechnology was used in a very early innovation that had outstanding potential impact, but was impeded by a process and by a misunderstanding and maybe a poor education of what it actually did and something that we should have been excited to accept got pushed away and never was actually implemented.
But this is a great rule of thumb because we're moving into an accelerating period of innovation where the only way we're going to possibly get these to reach the places they're meant to serve is by education and good communication. So this is the Talking Biotech podcast and we'll talk to you again next week. All right. And we will stop. Good.
Steven Earl Lindow (26:53.199)
Okay.