 at the Technische Universität Tresden in Germany. Her full title is Dr. Susan Auer, with two N for Susan, don't get the wrong. And the title of the talk is Molecular Response of Collaborative Effective Plans to endophytic fungus acrimonium alternatum. Thank you very much and please take it away Susan. Yes, hello everyone. Let's see if that's going to work. So I'm sharing my screen now and I hope you can see it. So how does it look like? Do you see what I see? Which should be a presentation. Yep, that's great. So, first of all, thanks for giving me the opportunity still, although there's some poultry, which is good to know. So that's pretty democratic, that's really good. Just a few words about me. So I'm a postdoc, but I'm mainly actually a lecturer right now. So I don't really have so much time for science. I just try to do with my students what I can. And I want to talk today about food, coverages and the molecular stuff that we've found out that the plant actually doesn't respond to the disease when it's also challenged with another fungus, which is acrimonium alternatum. So the first thing, because out of experience, a lot of people do not know so much about club root, I think I have to go a little bit into more depth here. A lot of plant pathologists also don't know the disease, probably, so don't feel bad if you haven't heard about it. However, a club root disease is distributed right now, actually worldwide. This is pretty scary. When I started with club root disease something like 10 years ago, this was not how the map looked like. It was like one quarter of it or something. So now a club root disease is everywhere where brassica plants are grown, because club root disease is specific to proper crops. So you can see you have it here all kind of on all continents so far. Some truths and hard facts about club root disease. Club root disease is one of the most damaging diseases in crucifer crops worldwide. So all kind of cabbage plants are affected by this disease. Boy seed rich, also called canola in Canada or rapeseed as I refer to it, which is a huge cash crop. Broccoli, cauliflower, all kind of cabbage varieties you can think of are affected by this disease. And you can see here a few, you can see, I'm not sure if you know, if you see my pointer, but you see here, so on the left side of these panels there's always the control plant. So this is a rapeseed and on the right you see these specific club calls. So the thing is with club root that it was described already actually a little time ago in 1878 or something. And we still don't know so much about that disease. And just pulled up some numbers. So I don't know right now what's the situation in Australia but or in Canada and Canada, it's pretty dire right now as well for the European Union. I have found that the estimated annual yield loss right is 700 kilowatts of a rate which jumped up to 630 million euros. And this is just for the year 2018. So this is a pretty devastating disease also economically. So if you look at rapeseed it's just one crop where this is affected. So this is really bad. Also it spreads actually tremendously. So in Canada they did some digging deeper into the numbers and they found that it started in around 2000. So they had in 2003 they had something like 12 infected fields and now they have over 1000 and that was only in a couple of years, a little more than a decade. So what's the problem actually with club root? I want you to take away if you take anything away from that talk at least three facts about club root. The first thing is that club root is caused by a traffic protest. That's called plasmodiorfobrasek. So this is a eukaryotic protest. You can see here on the right a funny little, well pretty colorful tree. And you see on the top the rice area. So the rice area are a group which have several groups in them and some of them are plant pathogens. So the phytonyxids, this is the group where plasmodiorfobrasek is in. You might know another species here from the plasmodiorforids actually which is spongospora subterranea which causes the pottery potatoes gap. So I have just this really, if you address this really, we said in the paper review, a lot of times the review is to refer to this as a fungus or something. But if you just look here at the bottom, so the fungi are here. So we have a protasekaryotic protest. It's not an omnisate, it's a protase and it's also not a bacterium and not a virus. So that needs to be taken into consideration when we talk about that disease. So it's a little specific and proxotic. The second thing I would love to have you know about is that plasmodiorfobrasek has a very complex biophasic life cycle. So this is in a way it's very interesting and it's also on the other hand, preventing us from combating that disease easily. So what happens is we have the resting spore you can see here on the top. The resting spore that is very durable and can stay in fact in soils up to 20 years. This resting spore hatches a sews spore which in fact the root hairs of susceptible plants and then in these root hairs it propagates into something that's called primary plasmodior and at least the second type of sews spore, secondary sews spores which then infect cortex of the root. And then we see these symptoms and the root swelling. So here's a microscope you can see in the middle and the top you see normally the root is as you know pretty well structured. You have the central cylinder and then you have all the other cells nicely organized around this. And what Clubroot does actually is it disrupts the structure. So because the root cells at some point filled with these plasmodior, which are the machineries for the resting spores in the end and they are filled with resting spores which really disrupts the cells and lets them break off. And it also very enlarged the cells tremendously. So this is why we have this root swelling. Third thing is so Clubroot is soil. So it stays in soil and it's an obligate bio-trouble. So that means it actually stays in, when you have a field that has been infected or that has been infested with Clubroot, you will have that field being infested for the next couple of years as well. So once you have it because when the plants break down they release these resting spores which I just mentioned, they stay infective in the soils. They can just any other brassica crop that's up to come. So actually a field that has seen Clubroot is not suitable for any more brassica cropping. And that's a problem. It's a problem because let's just take the crop rapeseed again. Rapeseed is huge cash crop use from rapeseed we use for human consumption as an oil or it's used for machine oil. It has a lot of different applications and farmers get a lot of money when they crop this. So Canada, for example, as a country depends a lot on rapeseed production because they export that. It's very important for the economy because they have really big problems with Clubroot. They took a lot of money in hand to fund actually some good science projects and a lot of scientists and they do very great research on Clubroot right now. Unfortunately, the good funding situation is not the case with a lot of other countries. So in the European Union, for example, each country kind of tries to it themselves and this is not really for the benefit as far as I. So what happens soil acid with these, we know that infested soil moves from field to field mainly with machinery. So a huge tractor, a huge field tractor can have something like 50 to 150 kilogram of soil on it that in worst case, spears these resting spores and that just get for two else. So this disease, not respect, country board is a situation we have now and that farmers are in the tight bounds, farmers are in, they have to make money and they don't really have the time, most of the time to clean their machinery really extensively as it would be necessary in order before they move to another field. So this is what helped to other fields. Control measures that people have undertaken are they use linings. So they try to raise the pH of the soil because we know that with a low pH Clubroot infects much better. Fungicides have been tested because Fungicide is an against zoos for different modes of effects. Sustainable would be to have a wide crop rotation to not have rustic crops for the next consecutive years but to have a break in between for five years. But none of these methods so far is sustainable. So a crop rotation is basically would be one of the best options sometimes but it's not because the farmers are lying on these cash crops in order to get their income. And what we also see is when we use chemical control a lot of that actually is not in the European Union anymore but the response to chemical control on Clubroot fields is very inconsistent. So now an idea is what works for a lot of other diseases to really use integrated pest management practices to have a combination of practices in order to control that disease. So this is just a little cartoon I drew to show you what kind of IPM tools would be available. And it's so that it starts with the outer layer where you have a preventive crop protection measure which would be crop rotation or using tolerant or resistant cultivars. And then the middle we are really at the direct treatment of the crop. So resistant cultivars of course are an option. However, what we know is that Plasmodiorthra develops within the fields, it comes in different pathotypes that have a variety degree of viral lands and aggressors and they have come all of them cultivars before. So the resistance is broken down by the pathogen because it favors specific very aggressive pathotypes and then these resistant cultivars are not an option anymore. So we are for this talk today we are here at the biologicals or the biocontrol agent which could be used in combination with other strategies in order to combat that disease. This is a little lap peak how we worked with Plasmodiorthra brasekame. So as I said before, I have to stress it again it's a biotrope so we cannot cultivate it outside of the host. And the full developmental cycle until you have the resting spores from the plants take something like four to 12 weeks depending on the susceptibility of your plant. So we use Arabidopsis for a lot of our studies because this is, well, you know the genetic resources are there and when we search for new breeding targets this is where we start our search because as I said before a lot of stuff is not actually known about Clubroot so this is, you have to start somewhere and here you can also, you can manage it a full experiment within six weeks. In brasekame, so I use, for example, rapeseed or a Chinese cabbage the whole infection cycle, the whole developmental cycle takes something like eight to 12 weeks before you can harvest and have these gulls to work with. And then we store the gulls in the freezer and extract those spores when they're ready. So the thing is you cannot work in exotic cultures with Clubroot because you always, when you do this spore extractions of the resting spores you always have a contamination with plant material that also has made the sequencing of that produce a little challenging. So exanic cultures are not possible so all the experiments that can go pretty quickly is not really possible with Plasmodiafra. So what I said here and that con is a Cromonium alternate, a Cromonium are pretty simple built fungi. They're very, one of the most simple structured filamentous anamorphic fungi you will find. And they are pretty ubiquitous so you will find them in a lot of different environments. There's known to be marine species of that and a lot of land and habitat species and they can colonize a diverse range of organisms. So there are papers of acrimonium species about entomopathogenic fungi. There's at least one paper from clinical research that reports about an acrimonium species that they found in a patient. Then there's a lot of plants colonized with these and also fungi so they can also hyperparasitize fungi and I'm pretty sure they can also colonize bacteria. Some of these acrimonium species reduce specialized compounds such as acrimines or acromolectones and the species I work with so far has not been reported to produce any specific compounds. However, the phylogeny of that species is a little complex as with a lot of fungal genetics and I'm using one particular strain that has been shown to have actually a good against some diseases. So the type of fungus I work with has been reported from the 90s from Brazil actually to work as a biocontrol agent against tar spot disease. However, because they don't report strain names or numbers, it's not completely clear if it's the same species, the same strain. We know from some European soils where you can find acrimonium alternatum because it likes to inhabit warm temperate soils that it has some effect against powdery mildew in various cultivars. And the specific strain that I use has been reported to reduce the feeding of diamond moth larvae in cabbage and it also increased flight of sterile content in these plants. So what I have tested so far with some colleagues is what it does colonize. So it works on rapeseed, it can colonize Chinese cabbage, maize, wheat, tomato, and then as I said before I use it in Arabidopsis a lot. You can see here, this is just the variable in our staining of Arabidopsis roots. So you see this very tiny hyphae which are pretty hard to see sometimes within the tissue. And then you can see here from tomatoes. So this is like a re-isolation experiment I did on tomato. You can see what the fungus here is growing out of this. So the type of experiments are, I use soil cultivation for anything it has to do with disease rating of club root because as I said before it takes something like four weeks until you can see the symptoms and assess the disease. And then I use in the middle, I came up with a hydroponic culture solution. So the thing with club root is it does not, in fact, as I said before, it can't take any culture dishes or anything. And actually the goal of development is best seen in soil. So in sand the goals do develop but not so good. But when I do, when I do RNA extractions and I do a lot of transcript studies I have to have a method to have a very easy access to the roots because roots are the main tissue that I investigate right now. So I came up with this hydroponic culture system. It's a combination of what other people have tried as well. This is also inspired by a paper that was growing broccoli in sand. So these are 10 millilitre pipette tips and they are filled with sand and then watered with a nutrient solution. They stand in that and here the hardness, these roots here is super quick and easy. So it's a matter of seconds. And if you just think about how much material you need for a good RNA extraction, not so much anymore maybe but when I started my research, something like 10 years back you still needed considerable amounts of biomass. And so this is what I use right now for a lot of my RNA extractions. And then to study the interaction of my fungus, acrimonyl, alternate with all kinds of plants I use a lot of exonic cultures. So what we study here, mainly as I said, is the pathosystem. What happens when we, in fact, Plasmodiophora with Clubroot is you will get these standard smaller plants here on the left, which have the typical Clubroot gauze with these symptoms and with microscopy you can see that there are some spores are forming in there. If you, in fact, the same Arabidopsis with acrimonyl-alternatum, you don't see any symptoms. That's very typical for a lot of endothelic organisms that they don't make any symptoms, no obvious phenotype. However, what I have noticed over the years is because I've done it a couple of times really is that the reproductive success of a lot of the plants is actually better when they are inoculated with acrimonyl-alternatum. So these plants will produce more flowers. There's also small paper that we published on that in Rape Seed. And when we combine both of these microbes, we will have a plant that does not suffer so much from Clubroot. So they will still get root gauze, the Clubroot gauze, but stems are longer. Reproductive success, the biomass is actually larger than in those plants that have been inoculated with Clubroot only. So before my research on that, we know already that acrimonyl-alternatum can suppress Clubroot disease. This has been done in the group I work in right now before my times, and this is what we are still seeing consistently, pretty much. So you see here on the right the actual plants. And also it has been shown on Chinese cabbage that an infection with plasmodiorfra and acrimonyl-alternatum actually decreased the infection rate of plants. So less plants were infected with plasmodiorfra. And the disease index, which is a measure of the severity of the disease, was lowered. And this is something we pretty much see consistently, which is really nice. So I look just to speed that up. I look at gene recognition in plant cells. So I do a lot of RNA extractions. I do a lot of QPCR before we go to the omics studies that we do as well. Here's just a small working model of the early response genes I look at. So you might know that when pathogens invade plants that they are detected by plants due to specific receptors. Here is just an example of two of these. So flakalins send FLS to receptor and then bakma, which can work together. And they can detect these so-called the microbial-associated molecular patterns that pathogens release. So these are conserved structures, which can be detected. So flakalin, the flakalin sensing receptor detects flakalin just as the name says. And then downstream of that recognition, we have a lot of other genes that are activated over certain plant hormones. Here for example, we have ethylene. So this recognition can lead to colostiposition and other measures that the plant can take in order to defend itself. So colostiposition is used to strengthen the cell wall. Then there is compounds produced, which have antimicrobial properties and so on and so forth. And what we also have simultaneously is the PTI response. So the pathogen-triggered immunity response that at some point in the cascade can activate a working transcription factors, which then can activate PO1 for example, which is the pathogenesis-related protein one. I think there's going to be another talk about pathogenesis-related proteins. And I'm looking forward to that. So this is the other response that I look at. And I also look at intermediate and late responses. So again, this is just a very small kind of model that highlights some of the genes that I used to look at in the last years. So from the side of the cellocytic acid, which is one of the major stress hormones and the genes that are triggered in that cascade, and then jasmineate and ethylene-related genes that are triggered, these are the genes that I look at. So this is what I mainly do. I look for dopses and a lot of these time points, intermediate and late time points. And in the end, usually there comes the disease rating. You're in mind. So there's always these four groups that I look at and they need a crucial amount of plans. So a lot of these transcript analyzes are done on roots, the major point of study that I had in the last course. And this is destructive. So there's always a lot of plans you have to raise, or you need a lot of hands, which we don't have actually in the lab. And then with Praseka, you can do similar things. And this disease rating, as I said before, takes a little longer until it's fully developed. So now just a few of the stuff that we found. We used a microarray a couple of years back to detect early responses in arabidopsis roots. And what we found was, first of all, that the numbers of differentially expressed genes in the mixed inoculation of acrimonium and plasmodiophora was actually much higher than, well, not much higher. It was higher than what we found with plants and plant roots that were only inoculated with acrimonium or only with plasmodiophora. So this was an early time point that was laying within the timeframe and plasmodiophora still gets established in the first primary infection stage where it makes these primordial, these structures in the beginning that release the primary sewer spores. So it could be that the few genes that were triggered here are actually because there was some sort of a lack of response in the plants because the infection of plasmodiophora was not established that well, but doesn't explain why we have a lot of genes triggered then when we have these covenuclein plants, which have here the acrimonium and plasmodiophora at the same time. So the response of plants to both of these microbes at the same time was elevated. That's something we see also with a lot of the genes we investigated. Sustain visualization of the stuff we found here at a very early time point. And you can see a lot of hormones are actually involved here in that stress response. You see cell wall changes. You see proteolysis, PR proteins are triggered. And then here we see signaling working transcription factors and others. And secondary metabolites are beginning to be triggered, but if they will actually produce remains to be seen. This is the comparison with other groups. You can see here, this is the plasmodiophora one, where you see not so much is going on. So less genes here are triggered and also again, here less genes are triggered. And there's not so much overlap between these three groups. So we did a lot of PCR also to confirm findings. And what we came up so far is that from the genes I showed you earlier, at least the genes for the receptors that could probably detect plasmodiophora or acrimonium or both. We saw that in this mixed inoculation, the fungus led to something like an increase in FLS2 receptor transcripts. So we've found more of this here in comparison to with plasmodiophora only. So if you look here at the top right and in the middle, you see actually FLS2 was not triggered at all in this early response. Some papers and some papers, it was speculated or actually it's one of the, well, one opinion right now is that probably when buck one and FLS2 work together, probably they can trigger that, they can of course make this pathogen triggered immunity response. And maybe they are also able then to help the plant to defend against club root. Because what we also found in later time points is that PR1 was actually triggered and the level of PR1 in the co-inoculated plants with plasmodiophora and acrimonium is always higher than it is with plasmodiophora alone. So obviously the fungus does something either it enhances some recognition or it does something else. So you can see here with acrimonium alone only FLS2 was not triggered actually. It was down-regulated a little bit and also buck one was only slightly up-regulated. But yeah, these are just some intermediate results right now. What we find consistently with all studies is always PR1. This is a very consistent response whenever we have a co-inoculation we have both microbes in the plant. We always have elevated on PR1 compared to the other two treatments. So the plants have in theory probably shown elevated response that might lead to these decreased symptoms to that suppression that we see in the end. We started to do some omics, some proteomics. So I'm just going to go through this rather quickly. We now look also at the upper plant parts at the shoot. So of course it's nice to have some transcript data with PCR or microarray where you would do probably direct RNA-seq today. With the omics response, however, of proteomics you can see what actually comes up then in terms of protein turnover. And here we found so in roots that yielded much more proteins, which is not surprisingly because you have the rupusco in the shoots which give a super large noise, so to say. And it covers a lot of things you probably may not attack then in shoots. So in the roots, we found some very interesting candidates. We found a lot of protein in their metapolar. Further look into this with functional analyzers. And just here are candidates which are interesting in terms of plant defense. So in roots we found an elevated amount of root endokitinase proteins. We found some other hormone-related proteins which were either more abundant or less abundant than in the other groups. And so far we found also some very interesting candidates which we will pursue further. So I'm not going to show that here now because it's still intermediate but we also, what we do now also, we look at the plus one you offer proteome actually to see what's going on in here. Then a few more words about rupusca. So I'm working also with seed. You can see here the symptoms of seed weeks after not killing. And on the right, you see that actually when if we think about priming, we want to introduce you, but that has an elevated defensive response when it is challenged with a pathogen that's not so drastic. And then it gets challenged again with another pathogen. It's actually pretty drastic, something like plasmodiorphera. You can sometimes see the weighted often actually plants combat disease much better. And this is what we tried here in rape seeds. So I first challenged the plants with the fungus and then I inoculated with plasmodiorphera. And in this small timeframe that you have in between both of these treatments, you could already see that the number of uninfected plants here on the right, on the top pedal on the left, the number of uninfected plants were considerably more than the number of uninfected plants that were in the control group with only the plasmodiorphera. So also this is just a hint, I mean, to elaborate on that, but use the fungus before we actually challenged plants with plasmodiorphera, we actually see the plants doing much better and have an increase in vitality. They have better reproductive success and they also have larger biomass. So the future paths we are going right now with certain different collaborations. We now luckily also have another postdoc in our lab who's working on global disease. We have industry partners that we work together with. We are now looking at that interaction from the omics part of you. So we're going to do proteomics, metabolomics, lipidomics. And then look at some hormone related transcripts. We started looking at cytokinin transcripts in the roots and then how that transpires into the proteome. And now we are also right now doing more work in acrimonium with some collaboration partners. However, if you're willing to collaborate on working on this specific fungus, I would be very happy to chat with you because as I said, we don't really have so many hands and I'm always eager to look for companions who work on that with us together. So that's it for now. Thank you very much for tuning in. Please stay in Zion. You're Zion's in a lab where no one will enter for the next weeks. As I said, if you have some collaboration ideas, I'm very open to that. Working with Clubroot can be fun, as you can see here. So we have these different phenotypes here. I dubbed them the chicken, the caterpillar, you have a great time and you don't feel isolated so much. So thank you very much. Who are gonna do the clapping? Thank you. Thank you very much. It was very interesting. Very good entertainment with wine. So there's a couple of questions and I'm gonna just pick some of those. So the first question which was there was about, so you just played the differential gene expression plus minus the fungus and then plus minus the bacteria, like, sorry, the protist. So what do you think the molecular mechanisms are there? Like how does the fungus together with the protist actually to enhance PTI components? So yeah, one thing is, as I said, I try to mention the maybe a better recognition. So when the fungus triggers genes that are used for recognition of pathogens, probably that's working better with acrimonium alternatum because what we found was, what other people also have found that you have a suppression in these very early detection mechanisms. So we know from plant immunity, if the plant's able to detect specifically some sort of pathogen, then it can respond. If it doesn't detect, then it cannot really respond accordingly. And if you disrupt, if you're a pathogen and you disrupt that fine-tuned machinery of recognition and response, then you can be very successful. Plasmodia fra also does a lot of effectors. So you can also, this is something that colleagues of mine are investigating right now. So it hasn't been so well investigated yet, but that's the second thing. So I think recognition, that's the thing. Probably acrimonium does also compete for resources. So there's something that just space could be because it colonizes pretty rapidly. So it could make also something like wraps a tight hyphen network and then the fungal, the protists cannot invade so well anymore. The sewers spores of the protists are actually a weak spot of that disease because they are short-lived. And if you, for example, that's what people have tried. If you can have the resting spores hatching and have these sewers spores in the soil and then you present it with some crop that can be probably infected but that does not develop the disease you can get rid of some of these club root contamination in the soil. So maybe it's a spatial thing or it's a recognition thing or it's, yeah, it plants strengthening thing. So different opportunities. There was another question which was along the protest. So, which I forgot the name already. So the question was, how many effectors do you have and how has these effectors delivered? I'm not a good person to speak about that. My Canadian colleagues are researching that. So if you're interested in that, the search for anything that Canadian's publishing right now, how they are delivered. So what the protest does is it actually injects its cellular content into the plant cells. So it works with, there's the German terms for this. I'm sorry, but it's called Stache and something else. So it actually does inject its cellular content. So this is the mode of infection for that protest of how it does in the plant cells. So the effectors also will have a secretion signal or the effectors also going to be delivered just by some sort of secretion, whatever. So if it's inside of the cell, it's anyway, if you release a factor that's not detected by any receptor, by any, I don't know, then you're pretty successful. Right. So there's another question just coming in and there were more other questions. So in the disease control perspective, do you think that acrimonium and inoculation would activate plant immunity continuously and would lead to plant energy waste when it's not infected with the protest? Plasmodiophora. Yeah, people call plasmodium also, it's also four. Plasmodium is a different disease. So the question was, if you use that fungus and by control, if it's going to have that, the disease or the kind of resistance, if it's up all the time, did I get that right? Like if it would lead to like a base of energy and so, but like, would it be like it meant to supply it? I asked myself this question a lot as well because as I said, so we have a better reproductive success, they have more flowers. Usually more flowers means less biomass, but if I measure the biomass, it's not actually changed that much. I don't know what the fungus does in or in terms of a resource allocation, but so far, of course, the thing is, if you have priming, I mean, priming is a very low energy response. It's very energy efficient. And I'm pretty sure that acrimonium is capable of doing that. So this would be not so detrimental for the energy metabolism. But the thing with the flowering thing and raising the resistance of the plant, of course, the plant will always have basically a fitness, well, it will have a problem with its fitness because it has to put some coast, of course, into development and others into defense. That's, I mean, that's always the trade-off thing or that's always the competition. I think it's capable. So what we see is the PR1 level, as I said, it's always higher when we have both of these microbes, but they rise throughout the course of the disease. And these plants are doing better against the disease. So they were probably in the end, if you have a field application, you might have smaller plants, that's possible. But the trade-off for that would be, you have a smaller plant that still is able to reproduce because for rapeseed, you want the seeds. So you have seeds, not so many, but you have them. In comparison to, you have a disease plant and you don't really have any seeds or the quality is really bad. So these are the two things you will have. The thing why we work in biocontrol is, or why I'm interested in that, in the European Union, there's a lot of the chemical control means that the Canadians can use or that I'm pretty sure you can use in Australia. They are not available to us because they are detrimental for our health and for environment and so on and so forth. So we have to go into that direction. Acrimonial alternation will not be the solution. It will be probably a combination of different methods and probably if we use that fungus, it could work. But yeah, side effects and stuff, we haven't studied that yet. All right, so more questions, if you don't mind. So there's a question around, like a more molecular question again, around, it's a relationship between PR1 and cytokinein and does the cytokinein effect explains the flowering biomass resistance trade-offs? So the first, the questions, the cytokinein explain the more flowering, yeah. Yeah, yeah, yeah, yeah. So we look into cytokinein response right now to see if that flowering is related to that, although we can look at ethylene as well. We found so far, but this is exanic culture stuff, the fungus is able to trigger IPTs. So these isopentymal transferases, which are responsible for cytokinein production, could be, so they are elevated, but actually it's a little complex, only under certain conditions. So under certain stress conditions, the fungus can trigger cytokinein related by synthesis genes, but it does not under other conditions. And the other question, I didn't understand because my internet was like not proof. The other question was about, is there a relationship between PR1 and cytokinein response? Cytokinein, I think right now, some cytokines are known to be involved in resistance responses. I'm not able to answer on that yet, actually. So we started looking at that, but yeah, no. There are a few papers about the relationship of cytokinein and defense resistance implants. They are better explaining that, I think. I'm not, in that interaction, I cannot say much about that yet. All right, so, and another question was, so are there any fungal secondary metabolites involved in the response, especially maybe suppressing the protests directly, look, as an anti-protex activity? That's very likely. The thing is in direct interaction against fungus, against fungus. Acrimonium is not very competitive. So against very aggressive diseases like phosarium or aspergillus, if you do just a normal interaction study, it just loses. So it's an endophyte, it grows super slow. I'm pretty sure it can do some specialized compounds or I'm not sure, it's very likely, but we haven't found them so far. Because as I said, it's not very competitive against other fungi yet. I haven't tested bacteria. What it is able to do is produce oxen. That's something we found. So it can make oxen. I mean, a lot of fungi, you know, can make hormones that are plant hormones, basically. Well, just some compounds in that case coming out of the tryptophanes biosynthesis. So it might be also able to produce a kind or something, which would explain the flowering thing. But for secondary metabolites, I have no clue yet. I would have to do some analytics to do that. And I'm not yet there. Time-wise, it's skill-wise. All right. I think that's all the questions I have for now. If people have other questions, you could always go to the Slack and ask Susan something or send her an email, of course, too. Thank you very much, Susan, for being our first presenter in this series, which we'll go on for some time. Hopefully also in the future beyond this. Thank you very much for being our guinea pig and having a great talk. All right. So at the end, it's the last day. So this was number one. We will have a seminar next week, which is by Remco Stam. This will be on Monday at same time. So 7 p.m., no, 7 p.m. Australian time, or 9 a.m. European, Central European time. The seminar will be entitled So Diversity, Molecular Evolution of Plant Defense Against Passage and in Nature. So stay tuned, come along and check out the OPPP Slack and webpage for more information because we already have three or four more seminars planned. And so there will be, you can also sign up via Google Form. So thank you again for Susan. One round of applause. And you all have a good day, good night, wherever you are. Thank you. Bye.