 This is defense, this is the public component. Carl joined my lab five years ago, and a little bit. We had a wonderful time here, and we're gonna miss him a lot. He's been a huge amount of fun to work with. I'm not gonna take anything away from Carl, because right now, I'll take any more time because I know he has a lot to cover. So, with that, it's all yours. Great, thanks for the introduction, Ivan, and thank you all for coming. It's nice to see all these faces, friends and family, and coworkers. So, today, I'm really excited to have the opportunity to talk to you guys about two of the projects that I did in the Raymond Lab, and they both center around the enzymatic inactivation of trichothesene mycotoxins in fusarium headlight of wheat, and we'll unpack that title throughout the presentation. I'll start out with a brief introduction into fusarium headlight and what it is, and then I'll talk about trichothesene mycotoxins, and then we'll get into the results, starting with the structure and mechanism of OS79, an enzyme that I worked with, the specificity of that enzyme, what I did to broaden that specificity, and finally, I'll tell you a little bit about how I developed a high throughput selection protocol to identify trichothesene inactivating enzymes. In 2017, wheat and barley were the second and fourth most produced grains in the world, and this probably doesn't come as much of a surprise given the vast number of food products that are made with wheat. This image from the USDA kind of highlights how, no matter what meal you're eating, you probably have a component that is made from wheat. Breakfast, lunch, dinner, even dessert, they're very common throughout your everyday life. Barley is also very important in a lot of food products, including a favorite here in Madison, beer. These are two of my favorite beers brewed at Carbon 4, just a couple miles away from this room right here. Unfortunately, production of wheat and barley is being threatened and has been threatened for some time by the fungal infection fusarium headlight. And this can be seen in this map right here where each dot represents 5,000 acres of wheat that were affected by the fungal infection fusarium headlight in the years between 2000 and 2010. The different colors represent the different cultivars of wheat that were affected. And these incidents of fusarium headlight cause massive economic losses for farmers. In fact, there was a two year period where farmers in North America lost $2.7 billion to this disease. And it's not just a problem in the United States, it's also a problem around the world, including in Europe, where every single year farmers lose 10 to 40% of their crops to fusarium headlight. It's worth noting that fusarium headlight is not just caused by one species of fungi, but rather many. And all these species of fungi can cause fusarium headlight and other related diseases and they're all in the genus fusarium. Fungal infection starts where they're all Ascomycetes and they rely primarily on Asco spores, which are ejected from the fruiting bodies of the fungus during the spring when rain and human weather causes the ejection. These Asco spores then land on the wheat heads and as soon as they enter the wheat heads, the fusarium fungi rapidly activate a host of genes that facilitate further infection. Some of the most important genes that are activated at this stage of fungal infection are the tri genes. And these genes are responsible for the biosynthesis of trichothesine mycotoxins, which are potent inhibitors of eukaryotic protein synthesis. And you might be taking a guess at why these toxins are so important for fusarium infection in the sense that if these fungi can produce trichothesine mycotoxins that spread ahead of the fungal infection and eliminate the ability of plants to synthesize proteins, they've eliminated the ability of the plant to respond to the fungal infection. So this leads to the spread of fusarium headlight throughout the wheat head. And that can be seen in this image where a wild type strain of fusarium that can produce trichothesine mycotoxins was used to inoculate a wheat head and after about, I think this is probably three weeks, the entire wheat head has been infected. There was a mutant strain of fusarium where tri-5 was disrupted and this strain is unable to produce trichothesine mycotoxins. And you can see the infection did not spread from the initial point of inoculation, demonstrating that without the production of trichothesines, fusarium species can't spread throughout the wheat head. And that can be seen in graphical form right here where the solid lines are a wild type fusarium and the dotted line is this fusarium species that cannot produce trichothesine mycotoxins and we have the percent of wheat heads that are infected and this is days after inoculation. So you can see again that the fusarium species that cannot produce trichothesines unable to fully infect the wheat head. At this point I'll talk a little bit more about trichothesine mycotoxins and there are a vast number of these trichothesines. Over 150 have been characterized and honestly there's probably many, many more. They're all characterized by this common trichothesine backbone, the skeleton right here, where the enormous chemical diversity comes from different chemical groups on these five R positions. And these chemical groups can be a variety of things including rather small hydroxyls as you can see in the valenol and deoxyne valenol. Up to acetyl groups as well as larger groups like this isovaluable group in T2 and HT2 toxin. I've included on this slide eight trichothesines in their entirety because these are trichothesines that I'm gonna be talking about throughout my presentation and I'll include the structures on the slides where they are relevant as well. As I mentioned before, there are many species of fusarium that cause fusarium head blight and related diseases and also there are many trichothesines and unsurprisingly that leads to a rather complicated arrangement here of where each fusarium species produces multiple trichothesines and in many cases fusarium species produce a different subset of trichothesine mycotoxins. These trichothesine mycotoxins not only help the fungi spread throughout the wheat head but they also accumulate in grain which is an enormous risk for human and animal consumers of that grain because as inhibitors of protein synthesis and eukaryotes they're very toxic to humans and animals. And again, these toxins can end up in beer because beer is made from barley in part and there was a study done in 2013 where they tested 374 beers from 38 countries and they found the trichothesine dawn in 93% of them which is a rather alarming statistic. Fortunately, the levels reported in beer in this study were no higher than 1.1 parts per million and the level that has been established as safe consumption by the United Nations is 0.5 parts per million. So they're still within the safe consumable dose however even consuming small doses of trichothesines is really not a good thing. These are the levels that have been reported in beer but crops of wheat that have been severely affected have shown levels as high as 20 parts per million which is of course significantly higher than the safe dose. It is lower than the 78 parts per million that has been showed as the LD50 which is the lethal dose essentially orally through mice. So at this point I've told you that trichothesines help the fungus spread throughout the wheat head and they're also toxic to humans and animals that consume that wheat. And so ideally what we wanna do is inactivate these trichothesines. There are two main ways to do that or categories really. The first is modification at this R1 position either through glycosylation or acetylation or other modifications. And the second is de-apoxidation and this critical epoxide bond. Now it's thought that these modifications disrupt the binding of trichothesines to the ribosome thus eliminating their ability to inhibit protein synthesis. The first project that I'll be talking about today looks at the glycosylation of trichothesines and the second project that I'll be talking about is aimed at identifying enzymes that can break this epoxide bond. We'll go back to this slide very quickly and I'm gonna add a little bit here. If we wanna inactivate these trichothesines one of the ways we can do it as I said is glycosylation at this position and an enzyme that's capable of doing that is a UDP glucosaltransferase where a glucose moiety is transferred from UDP glucose onto the trichothesine mycotoxin. If you express an enzyme that's capable of this reaction in wheat, hypothetically you would get a wheat crop that's resistant to the spread of fusarium infection. And there in 2012 a group did this and that's exactly what they found. They expressed a UGT from barley which is capable of glycosylating the trichothesine don at this position. And the fusarium, they infected that wheat crop with fusarium while type wheat crop was able to be infected with the fusarium species and the versions of the wheat that were expressing this enzyme were unable to be infected. At least the infection did not spread. And at this point you might be thinking problem solved. If you express the UGT in wheat, fusarium fungus cannot spread and you no longer have a problem. Unfortunately, as I mentioned, there are many, many species of fusarium and they produce a variety of trichothesines. And this UGT, this enzyme has only been proven to glycosylate dawn and a couple others. And so it only provides resistance to fusarium graminarum. And it does not provide resistance to other fusarium species that produce trichothesines like T2 toxin and others. One way around this is to look for additional UGT enzymes and hopefully you'll find one that has a broader specificity that can glycosylate more of these trichothesines. Unfortunately, that has proven very difficult using the primary sequence of these enzymes. And so structural data is required to do that. So the first project that I'll be talking about aims at understanding the mechanism and specificity of a trichothesine UDP glycosyl transferase and then using these data and the structural information to use rational design to broaden the specificity of that UGT. The first thing I'll talk about is just really diving into gaining as much structural information as we can about this enzyme. And then I'll talk about the second part. In order to do this, we solved the structure of OS79, which is a trichothesine UDP glycosyl transferase from rice. And this is the first structural information on an enzyme that is able to glycosylate trichothesines. So it was a very exciting first step for the field. And this enzyme is composed of an N-terminal acceptor binding domain, which is consistent of a six-stranded parallel beta sheet surrounded by nine alpha helices and a C-terminal donor binding domain consisting of seven parallel beta sheets and nine alpha helices. And you can see that these two domains come together to form a globular protein where the substrates bind in the middle cleft of these two domains. UDP is bound, unfortunately, there is no trichothesine in this structure, which is a problem for us because we want a substrate to be bound in order to be able to gain structural information about the specificity of this enzyme. In order to solve this problem, I co-crystallized OS79 with the non-hydrolyzable substrate analogs, UDP 2-deoxy-2-fluoroglucose, which is this right here. And this is just UDP glucose, except that at the two position, instead of a hydroxyl, we have a fluorine atom. And I also co-crystallized with trichothesine, which is the trichothesine, it's confusing, this one is actually called trichothesine, and it is lacking a hydroxyl group at the three position. And so you can see the glucose moiety cannot be transferred in this reaction. And so the substrates are locked into the active site of the enzyme. You'll notice that very good electron density allowed the unambiguous placement of UDP glucose analog in this case. However, this is a difference map of electron density for the trichothesine substrate, and this represents half occupancy. In addition to that, it's really just not great density. That, combined with the fact that this substrate is really kind of just a hydrophobic ball, made placing the substrate in a position that we were confident in rather difficult. Luckily, the position that fit the density best did make chemical sense, where if there was a hydroxyl here, it would be in line for an attack on the anomeric carbon. We did model that hydroxyl onto the substrate in order to come up with a proposed mechanism for this enzyme. And this puts the hydroxyl 2.7 angstroms away from the anomeric carbon at C1 of the glucose, and 3.6 angstroms away from a nitrogen on histidine 27. There had been structures already published of other plant UGTs that glycosylated other substrates, other than trichothesines, and they also had these two residues and aspartate in this position and histidine in this position. And that, combined with the fact that when we substituted these residues out for alanines, the enzyme did not have any activity. This led us to propose that this was the catalytic dyad for the catalytic mechanism. Our proposed mechanism starts with the deprotonation of the hydroxyl by the nitrogen on histidine 27. The resulting nucleophilic oxyanion attacks the C1 carbon, resulting in trichothesine with the glucose moiety and the release of UDP. You'll also notice this 3.9291 residue that I've highlighted here. This is 3.5 angstroms away from the beta, the oxygens on the beta phosphate of the UDP, and it's a pretty far distance for a hydrogen bond, but we think there might be an interaction there. And this residue has not been reported in any other plant UGTs, regardless of what substrates they act on. There's really two roles that we thought that this residue could be playing. The first is perhaps it could be playing a structural role to position the phosphate for attack. The other is it could potentially be acting as a catalytic acid, protonating one of these oxygens for UDP release. Given the very high KM at this position, or it's probably very high, any protonation would probably have to do with solving exchange of protons. In order to investigate what the role of this residue was, I overlaid my structure of OS79 and yellow with the six other plant UGTs that had been solved and deposited in the protein data bank. And you can see that UDP overlays quite well and is in the same position in all of these enzymes. And all of these enzymes have a hydroxyl at this position. It's not necessarily from a 3-ening, some do have a searing there, but either way they have hydroxyl that's within a hydrogen bonding distance of this oxygen. This confirms to us in a way that it's an important residue and in order to see if it played a structural role, I substituted out this 3-ening 291 for a valine which maintains a very similar shape and size at that position but eliminates the hydroxyl. I solved that crystal structure and I found, and this is it right here, so you see a valine instead of a 3-ening at that position. And you can see that the phosphate of the UDP takes a very different confirmation where instead of being upright in this crystal structure, it's rotated down in this crystal structure and this is the difference electron density that allowed us to place that phosphate. That can also be seen again where I overlaid the wild type and the T291V structure and you can see again that there's a very different position of the phosphate. This allowed us to conclude that this residue, at least in part, plays a structural role in positioning the phosphate for attack. And this could be really important for specificity because if there's such a stringent placement on the phosphate, it may have an influence on where the trichothesine can be located in the active site in order for a productive reaction to occur. With this part of the study was wrapped up, I really wanted to look at the acceptor binding pocket in more detail. Unfortunately, as you remember, I wasn't really confident in the placement of the trichothesine substrate. It was the best that we could do but I wanted more information there before proceeding. And so I co-crystallized OS79 with the product D3G and that's when Don is glycosylated, the trichothesine Don by OS79, which is a reaction that takes place, you get Don with the glucose moiety at this position. That is represented by this model and this density. You'll notice that the glucose moiety should be right here and there is no density for it. This is likely because the glucose has a large amount of flexibility on this molecule, which results in the density just not coming in in the crystal structure. This density represents 100% occupancy and although it's not the best, it does allow me to pretty confidently place the Don portion of that molecule in the active site. So I was more confident about where I had placed the substrate at this point. So I decided to look more in detail, as I said, at the acceptor binding pocket. One of the real features about this pocket is that it is a small hydrophobic pocket within the enzyme. This is electric static surface representation of OS79 where blue is positive and red is negative and you can see that it really is a hydrophobic pocket. On the left here, I have highlighted some residues that are surrounding that pocket and I looked at many of these to see what role they play in substrate enzyme interactions by substituting for different residues of these positions. I'm gonna tell you about four of these that I did, which I've highlighted here, these four residues and in order to do that, I used a kinetic assay that allowed me to track the rate of this reaction. So this is the reaction that I care about and I want a way to track that rate. And in order to do that, I tied this reaction to the drop in 340 nanometers that is accompanied by the oxidation of NADH to NAD+. And I coupled it through these enzymes, which aren't really important here. The important thing is that I can have a continuous readout of this reaction. With that continuous readout, I generated KM and K-CAT statistics for each of these enzymes and I did this using a Michaelis-Metton saturation curve, which just as a reminder to you who haven't taken biochemistry in a little while, this is a graph where we increase the substrate concentration, in this case, DAWN and I measure the rate of the reaction using that enzyme system that I just talked about and as we increase the substrate concentration, we approach the Vmax, which can be used to calculate the K-CAT of the enzyme, which is really the fastest rate of reaction when substrate is not limiting. The KM can be calculated by taking half of the value of the Vmax and it's at that substrate concentration that the KM exists. And just as a reminder, the lower the KM is, the better the interaction between, generally speaking, the better the interaction between that substrate and the enzyme is and the higher the KM is, the worse it is. So the first residue that I looked at was this phenylalanine-199 and this residue is about five angstroms above the hydrophobic backbone of the trichothesene substrate and I was wondering if the hydrophobic nature of this residue was important. So I substituted it for a glutamine, which introduces polarity at that position and this enzyme demonstrated no activity using my assay. So this confirms that this residue plays an important role in establishing the upper hydrophobic limit of the acceptor binding pocket. It's also a residue that is conserved in all enzymes that are able to glycosylate trichothesenes, which adds to my confidence that it is an important residue in the active site. The second residue that I decided to look at was glutamine 202 and when I looked at the crystal structure that I had solved, I thought that at 2.3 angstroms and a pretty favorable angle there that there was probably an important hydrogen bond between the don substrate and glutamine 202. So I substituted this residue out for an alanine and it turns out I was wrong. There's no important hydrogen bond there, at least probably not. In all of these tables that I'm going to be showing, I'm going to include the Km and the Kcat and the catalytic efficiency of wild type don, of wild type OS79 with don as a substrate on the top row and I'll change the values on the bottom row as I substitute different residues so you can compare them. So here you can see there's really no difference between these values of wild type OS79 and the Q202A substitution. It's also worth noting at this point that when we're talking about kinetics, in order for there to be a really significant difference, I really want to see an order of magnitude difference. You could talk yourself into a little bit less than that, but certainly 61 to 77 is considered no difference. What I would consider a significant difference is the glutamine 202 to a glutamate and in this case, there should be a negative sign right here. So in this case I was wondering if they're introducing charge of this position and had an effect and so I made the glutamate substitution and I saw there was a drastic increase in the Km which indicates again that there's a less favorable interaction between the substrate and the enzyme when this glutamine 202 is a glutamate. The last substitution that I made was a leucine and the reason I made this was I saw that there was no difference with the alanine. I was wondering if maybe a water had snuck into the gap that I had made and there was a hydrogen bond being made with the water. So I put a bit of a larger residue in the leucine at that position and to my surprise, this was the only substitution that actually dropped the Km. So this enzyme with the Q202 substitution actually likes dawn as a substrate more than wild type dead which is an important fact and I'll come back to that later on in my presentation. There were a couple residues that I substituted that really had no effect and that really indicates their second sphere residues not directing directly with the substrate. The first of these is serine 203. It's about 6.7 angstroms away from the substrate and I knocked it back to an alanine to see if the polarity of that position was important and it wasn't. I also substituted in a leucine at that position and I did see a significant increase in the Km with this substitution. What I really think is going on here though is that because of how important this Q202 position is I think that a leucine at this position disrupts the productive interaction between the substrate and the Q202. Alanine 384 is about five angstroms away from the closest carbon of the substrate and that again had basically no effect on the Km or Kcat. So at this point I had gathered a lot of structural information about the acceptor binding pocket. I had proposed a catalytic mechanism for this enzyme and I wanted to see which of the diverse group of trichythesines this enzyme could glycosylate and which ones it couldn't. So I tested the eight trichythesines that I showed in the beginning of the presentation and I found that OS79 could glycosylate these four but could not glycosylate these four. And this was really fun, it's like a puzzle. Why can't OS79 glycosylate these while it can glycosylate these? And if you look for a little while you can notice that all of these substrates, all of these trichythesines have an acetyl group with a C4 position and none of these do. And in many cases the only difference between one of these substrates like Navalanol and for a Navalanol is this acetyl group. The same is true between HT2 toxin and T2 toxin. And so I hypothesized that OS79 could not accommodate a trichythesine substrate that had an acetyl group at the C4 position. I wondered why this was the case. Luckily I had structural information to give me that answer. And so I looked at the structure that I had solved and this is a surface representation of OS79 that I've sliced down the middle. And you can see this is the C4 position, this is isotrichodermal and this is T2 toxin, one of the substrates that has an acetyl group at that C4 position. There just isn't room for an acetyl group in this structure. So I thought, okay, we'll make some substitutions to this enzyme to increase the size of the acceptor binding pocket in this region. And so I did that and I solved that crystal structure. In this case I made substitutions to histidine 122 which is this residue right here. Lucene 123 was this one and then also the Q202 residue that I talked about. Also the Q202. So here this is a surface representation of the enzyme with the substitutions that has the larger active site. And I've modeled back in these residues from the wild type enzyme. So you can see where they would be positioned. And looking at this, it seems like there really is room for the acetyl and there's also room for a variety of other groups on these positions. So I tested this enzyme with my assay to see if there was activity and I was really, really excited to see that there was. This double alanine substitution at these two positions was able to glycosylate T2 toxin. And this was a huge step forward for us because this is the first UGT that was confirmed to glycosylate T2 toxin. And T2 toxin is a really important trichothesine that's produced by fusarium spore trichoidies, which is a fusarium species that's really a problem over in Europe. I did make the glycine mutation first instead of the double alanine. This enzyme was actually really poorly behaved. And I believe it was because a glycine at this position was introducing too much flexibility to that loop and the enzyme wasn't folding properly. So I made the less drastic substitution of an alanine and that enzyme was much better behaved. At this point, I knew that it glycosylated T2 toxin, but I wanted to also know if it glycosylated other substrates that had the acetyl group at the C4 position. And so I tried it with these substrates and I found that it could indeed glycosylate DAS, fluorine, evalanol, T2 toxin, as I already said. Unfortunately, when I tested this enzyme's activity with dawn as a substrate, the KM had risen drastically from the wild-type enzyme. And this is a step backward for me because I want this enzyme to glycosylate T2 toxin and all the other ones with acetyl groups, as well as dawn, and I wanted to do it relatively well. So you'll remember back, there was one substitution that decreased the KM with dawn as a substrate that I had made and that was the Q202L substitution. So instead of using the A substitution, and these kinetics are just the ones from the slide before, I bumped them up, I introduced the Q202L substitution and I was really happy to see that this enzyme had a much lower KM with dawn. It's still not as low as wild-type, but it's significantly better than the 1202 that I was seeing with the first variant. So at this point, I had created an enzyme that could glycosylate all the trichothesenes that we tested with it and it could do it relatively well for all of them. This was a huge step forward for us because we've essentially created an enzyme that is capable of, or has the potential to provide resistance to all fucerium species and not just a few of them, no matter what trichothesenes they rely on to infect wheat and barley. Given the potential huge economic benefit that this enzyme has for farmers, the Wisconsin Alumni Research Foundation patented this enzyme. They submitted the patent, the regular US patent a year ago and we're hoping that in the future it can be used to create a transgenic crop that will have resistance to all fucerium species. So just to summarize, as I said, I generated lots of structural information about OS79 and this structural information can also be used to potentially find other UGTs that are able to glycosylate trichothesenes. And then I brought in the specificity of that enzyme to glycosylate all trichothesenes and not just these four. So that was the project that dealt with glycosylation. Unfortunately, glycosylation of trichothesenes is not a permanent way to inactivate them. It's perfectly good for stopping the spread of infection within the wheat head. But if you have already infected crop that's already harvested and you're gonna eat it, glycosylation is not gonna help because that glucose moiety can be removed in your gut which restores the toxicity to the humans and the animals. And so in order to be able to treat already harvested grain that's been infected, we would really love to have an enzyme that's capable of breaking this epoxide bond. There are no enzymes that are capable of doing that that have been identified at this point. And so one of the methods, one of the very promising methods to develop such an enzyme is to take an enzyme that exists that hydrolyzes a tertiary epoxide bond in a substrate that's very similar to trichothesenes and then making a large mutant library with that enzyme as a template. And hopefully one of those variations of that enzyme will be able to accept trichothesenes as a substrate. Creating the large library is really kind of the easy part. It's creating a selection protocol that can identify enzymes with the ability to hydrolyze this epoxide bond that's the difficult part. And so that's what I tried to develop. And it's a method to identify these enzymes. The first method that I tried was a pretty simple plating assay where I took one more thing. It's worth noting that throughout this method of development, for my positive control, I used an enzyme that acetylates trichothesenes at this position, which, similar to glycosylation, reduces their toxicity to eukaryotes. And as my negative control, I used an empty plasmid. So this is a pretty standard plating assay where you would take your gene library with your millions and millions of variations of this enzyme, transform a yeast strain with those, recover the yeast strain, plate them out onto minimal media plates, allow colonies to grow, transfer the colonies to a plate including the trichothesene isot, and then incubating them for three days. And the idea here is that any colonies that have a plasmid that contains a resistance gene will be able to grow on the plates containing the trichothesene isot and the ones that don't have a resistance gene won't grow. So I can just pick the colonies that do and problem solved. As many of you know with method developments, nothing goes according to plan the first time. And this is no exception. On my, after three days of incubation, on my negative control plates, I saw hundreds and hundreds of colonies. This is obviously a really bad thing because I only wanna see colonies on my positive control indicating that only colonies with a resistance gene grow. Colonies on my negative control indicate that yeast are able to develop resistance to trichothesenes independently of a resistance gene that I've added. And that's a huge problem. So in order to overcome that problem, the first thing that I did, I looked in the literature and I asked myself, why are yeast so good at developing resistance to trichothesenes? And I found two things. One, they include an acetyltransface in their genomic DNA that is able to acetylate some trichothesenes, which is my positive control. So we're already off to a bad start. So I deleted this gene from the genomic DNA of yeast and got rid of that. That shouldn't be a problem anymore. And then in addition to that, several papers identified mutations that occurred in yeast in the RPL3 protein of the ribosome. And the RPL3 protein is the closest protein in the ribosome to the position where trichothesenes bind. And so these mutations are occurring in that protein, which basically allows the yeast to be expressing or putting together a version of the ribosome that trichothesenes cannot bind to. And this is a problem because I want my yeast to be sensitive to trichothesenes. So in order to overcome that problem, I introduced, well I added the RPL3 gene to a plasmid, and this is a high copy number plasmid. There are about 30 copies per cell. And then I transformed my yeast strain with this plasmid. So now there's 30 copies of that gene in the yeast cell. So if the genomic DNA version or even a couple of the copies in the plasmid gain these mutations that provide resistance to the yeast, it's not gonna be a problem because the vast majority of the protein there is still gonna be wild type, which trichothesenes can bind to. For this, then the rest of the assay is the same. Unfortunately, I didn't get any colonies on my positive control here, or there were fewer than one colony per plate. And this is the problem because one colony per plate's fine if I wanna plate out under a thousand plates. And that's just not practical though. And so to overcome this issue, I decided to add a liquid selection step where hopefully I could increase the proportion of the actual cells in the culture that had a resistance gene. So that's what I did. And the old protocols on the top, and I've added the updated protocol on the bottom as I'm gonna slide it before. And in this case, after I had transformed the yeast with my positive and negative control, I added isot to 15 micromolar in the culture. And then I incubated overnight at 16 degrees C to allow the expression of any resistance proteins. And then I played it out on isot plates and incubated for three days. And in this case, I saw a bit of a better result. Here there were about, I think it was like 25 colonies around on average per plate on my positive control, which was a really good sign. And there were no colonies on my negative control. And so I introduced a couple more controls here. And you'll remember that really this selection protocol, I would love to be able to use to screen a library that has millions of variants in it. And so this selection protocol should be able to pull out a resistance gene, even when that plasmid is outnumbered a million to one. And so that's what I've done here, where in this control, for every one positive control plasmid I have, I have 10,000 negative control and one to a million here. We got a few colonies on this control, but none on the one to a million. And that's my benchmark. I want the one to a million to pull out colonies. So I needed to increase the efficiency of my liquid selection step. And in order to do that, I decided to add a stress step. And this is based on the finding that when exposed to stress, yeast change the expression profile in the cell. And these data are green represents fold repression and red represents fold activation. And there are several stresses across the top. And you'll see that the stress salt, and this is literally just NACL, table salt, increases the expression of lots of genes. And so I decided to choose this as my stress that I was going to introduce to the selection protocol. And I'm just gonna walk through this and kind of logic behind it. Where I have my, this is this, I added stress to these cultures. So I grew the cultures up after I transformed them with try one-on-one positive control and empty plasmid. Then I added the salt and I incubated it for three hours. Then I took portions of this culture and I plated them out to see what the cell density was in those cultures. And you can see the cell density was the same whether I had a resistance in there or not, or whether I had added stress or not. So this did nothing to the concentration of cells in these cultures. At this point, I hypothesized that cells without a resistance gene were gonna be very, very susceptible to any fungicide because they were unable to adapt to the stress because they couldn't synthesize proteins and the ones with the resistance gene could. And so I added amphotericin B, which is a fungicide to these cultures. And I found that was the case. Amphotericin B preferentially kills yeast cells that do not have a resistance gene that have been exposed to stress. And that was my key. And so I added this step to my selection protocol. And as Megan pointed out when I gave my practice talk, I couldn't have added another step because there's not enough room on the slide for it. So this better be the last version of the protocol. So here, it's the protocol that I used before on top. I transformed my yeast strain. Then I incubated overnight to allow the cells to express any resistance proteins. I added the trichothesene, ISOT, incubated for an hour to let that trichothesene enter the cells. Then I added my stress incubated for three hours, amphotericin B, incubated overnight, and then I played it out. And I was really excited to see that this actually worked really, really well. Even when my positive tri-101 plasmid was outnumbered a million to one, I still got hundreds of colonies on those plates after three days of incubation. So at this point, I had successfully developed a protocol that I or other scientists could use to select for genes that provide resistance to trichothesenes, which is a really, really powerful tool. And I'm hoping that scientists in our lab or else who are able to utilize this and develop an enzyme that can do that. As a summary of the research talk in general, I started out with structural information of OS79, the UDP Glucosal Transface that can glycosylate trichothesenes. I broaden the specificity of that enzyme. And as I said, this enzyme was patented and can hopefully be used to provide resistance to a wide range of fusarium species. And then I developed a selection protocol to identify enzymes that can break the epoxide bond, which can be used to treat already harvested grain and eliminate the trichothesenes in those grains. And that is reflected in my future work or future work that will be done on this project at least, where I hope that a transgenic wheat crop can be generated or a barley crop expressing this enzyme and having resistance to many fusarium species. The structural data that I gained can also be used to identify additional UGTs that are capable of glycosylating a wide range of trichothesenes, hopefully. And then the selection protocol can be used to identify an enzyme capable of hydrolyzing the epoxide bond. And Ivan made a couple of things clear to me before I joined his lab. One of them was, he said, Carl, I expect my grad students to come in and work their butts off and then be able to go home and have a life after. And that was really important to him and it was really important to me. And I'm glad that he had that philosophy and I couldn't have done that if it weren't for the amazing group of friends that I've had here in Madison that have supported me and all the adventures that we've been on that have made coming into lab and doing the science that much more exciting and meaningful for me. From going on hiking trips with my roommates around here in Wisconsin and also not in Wisconsin in Colorado, sailing with Tyler and Marcus and Alex, my amazingly supportive girlfriend and of course my family. I won't bore you with all of the amazing thing and fun things we've done together but thank you guys so much for an amazing five years. I really appreciate all the support that you guys have given me. With that I will thank the Raymond Lab members, my thesis committee, everyone on the Holum Lab, our collaborators and the people down at Argonne for structural work that they helped us do. That'll take questions. Questions from the audience? Yeah, great. Thanks for the question, Sam. So the question is once we had an enzyme that was capable of breaking the epoxide bond how would you administer that to crops in order to prevent the fungus? That enzyme wouldn't be administered to crops before they're harvested. Ideally really you'd have the UGT that adds the glucose and you'd have a transgenic crop that the infection wouldn't spread on and then you really wouldn't need the de-apoxidase enzyme. Unfortunately making transgenic crops is hard. People don't like GMOs and so that project could come into some hurdles and if it does the backup would be to treat already harvested grain. And in that case I'm not sure what they would do but incubating the grain with bacteria maybe that expresses the de-apoxidase enzyme wouldn't activate them. You could even sell a little bit of the enzyme and pop it in your beer before you drink it. I can see that taking off. It's a startup idea guys. So I'm not sure but there's several ways you could do it. You wouldn't want to purify the enzyme on mass though because that would probably be pretty expensive. Sam? Yeah thanks for the question Sam. The question is when we expanded the active site of this enzyme to accommodate the acetyl group at the C4 position right here are there any substitutions here that are bigger and would this enzyme would cost like those? I don't, there's hundreds of these trichothesenes so the very well could be them out there. I don't recall seeing anything bigger than acetyl group at this position. And if there was, it depends how big but I'm not sure the enzyme would be able to accommodate it. Absolutely, thank you, that's a good question. So the question is instead of just knocking these residues back to alanines which are small but hydrophobic given that the acetyl group is polar we could maybe introduce a residue here like a serine that could form a hydrogen bond there which would drop the KD even more because it would be a much more favorable interaction because of the energy that you gain from the hydrogen bond. I kind of thought about that and the real answer is that, well I could have tried it however I think that creating that specific interaction is much more difficult in an active site than just making it bigger. The residue would have to be very precisely positioned angles and distance wise for that to be a productive interaction. It could be worth trying definitely and that would be really interesting to do. I think your chance of success is relatively low though. Mike, yeah, thanks for the question Mike. The question is when I substitute out the hydroxyl containing threonine with the valine is this enzyme catalytically active? And I'm really glad you asked that because what I should have said on that slide is no it's not catalytically active and I meant to say that but skipped over it. So this enzyme is catalytically dead, yes. And this residue of course as I mentioned is present in all these plant UGTs. None of the papers with those structures have identified this residue as being important. So this kind of is a little side discovery here that's kind of unrelated to glycosylane trichothesenes and more related in general to the mechanism of plant UGTs and honestly potentially many other UGTs as well. Yeah, that's a great question. So the question is these UGTs, OS79 from rice and the other one from barley, are they in plants to provide resistance to fusarium? And the answer is perhaps in part there are many, many, many of these UGTs. In fact, in plants one to 2% of the genes in the plant can be UGTs and they glycosylate a wide range of substrates and glycosylation in that case affects by availability and localization and all sorts of things and in general their specificity is rather broad and so they can take on a bunch of different substrates. So it's possible that some of them are there and have remained there through evolution because they glycosylate trichothesenes but it certainly isn't their only job and there is no evidence to suggest that that's specifically what they're for. There are many that are up-regulated when you infect so that's one way they try to identify them is they infect the wheat head and then they see what genes are up-regulated and they've identified a lot of UGTs that are up-regulated. Almost none of those glycosylate trichothesenes which makes it difficult but at least the plant kind of puts them in that category of we have an infection, let's up-regulate a bunch of UGTs and see if one of them does the trick. Yeah, and that would be, so the question is could you add a bunch of these UGTs to a crop to over-express them instead of just one and the answer is you could. I am not an expert on creating a transgenic re-crop but I have read a little bit about it. I know it's very, very difficult. There are hexapointy, there's six genomes there, there's lots of transfer, plant genetics is just weird. There's lots of transfer and as of last year there's not a single wheat GMO that is commercially available and it's not for lack of trying. I'm sure Monsanto's been on it for a while but so I think that asking them to do three might be a little bit difficult. Delia? Yeah, so I'm not exactly sure what the trichothesene concentration in plants is. So I don't really know. But the KM is pretty high as you probably noticed from these. So it's possible that you need a really high local concentration of the toxin for a productive reaction to take place. Yeah, Hailey? The question is do I know how a species kind of decides or evolves to make a certain trichothesene or whatever substrate it creates? And the biosynthetic pathway, I might have a slide of it, is the same for most throughout, as you would expect throughout, yeah. So it's the same all the way up until some steps and so you really don't have to change much to change the trichothesenes that you make. And I do, so I don't know how really they decide. I do know that they can change though which makes it really difficult. There have been reports where certain fucerium species have actually changed the trichothesenes that they rely on over like five year periods which makes it really difficult. You know, that's why you don't just want a UGT to glycosylate one because they've demonstrated the ability to overcome that pretty quickly. You want your UGT to glycosylate all of them. And so I hope that answers your question a little bit. Okay, let's see.