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Welcome, everyone. "Wednesday Nite @ the Lab." I'm Tom Zinnen and I work here at the UW-Madison Biotechnology Center. I also work for UW Extension Cooperative Extension, and on behalf of those folks and our other co-organizers, Wisconsin Public Television, Wisconsin Alumni Association and the UW-Madison Science Alliance, thanks again for coming to "Wednesday Nite @ the Lab." We do this every Wednesday night fifty times a year. Tonight it's my pleasure to introduce to you Jean-Michel An. He's a professor in agronomy here at UW-Madison. He was born and raised in Montpelier in France, went to undergraduate at the University of Leon in France, and got his PhD at the University of Toulouse in France. (laughter) Yeah. Then, he did a post-doc at, I think we pronounce it UC-Davis? (laughter) And then came to Madison in 2004. He is just the latest in a long line of rather extraordinary researchers on soil microbes' interactions with plants. So when you think of people like Robert Burress and Ira Baldwin and E.B. Fred, that's kind of like the Curly Lambeau and Lombardi and Nietzsche of the world of nitrogen fixation. So, to let you have an idea of what Wisconsin has meant to the world from nitrogen fixation point of view and plant root microbe interactions, this is pretty cool. I'm looking forward to this. I was born and raised as a plant pathologist, and to get to hear about this, the symbiosis that are both helpful and hurtful, is gonna be great. Please join me in welcoming Jean-Michel Arne to "Wednesday Nite @ the Lab." (applause) Thank you. It's a pleasure for me to be here and to have the opportunity to talk about my favorite topic which are these beneficial associations between plants and crops in particular and microbes. So, the way I'm gonna structure my talk, I often use that image of a tree, as you can see, where I talk a little bit at the beginning about what we call symbiosis, what we call symbiotic associations, because that term is often the source of some kind of misunderstanding. And then as you can see, I'm gonna talk on the right about what I call the problem, which is that kind of fertilizer that we use, and I'm gonna talk about both the UT and the advantages of fertilizers, but also the problems that they cause, both in terms of sustainability of our agriculture and in terms of development for the developing countries. I'm gonna talk about microbes and what microbes can help, can do to help us solving these problems. That's the root of my tree. I'm gonna talk in microbes in particular, and all that, leaving the soil, and then I'm gonna talk about the trunk, the core of what we are doing in my lab as well as many other colleagues are doing in the world to try to better study these associations in order to use them. And that's really where I'm gonna talk about what I call the sweet talks and the trade deals. The sweet talks, it's a way for us to refer to the signal exchanges on how plants and microbes recognize each other, how a plant is about to recognize a beneficial microbe from a pathogen, for instance. The trade deals, it's about the nutrient exchanges. Plants and microbes exchange nutrients and these nutrient exchanges are highly regulated. In particular what the plants want to do is avoid cheaters, and I'm gonna talk about how the plant can avoid microbes that try to get benefits from the plant without giving it any benefit, anything else in return. And then for the fruits of the research, that's the application of the research. I'm gonna talk about what we do about relatively short-term applications, the low-hanging fruit if you want, what we do with serving farmers, in particular, also tomato growers in Wisconsin. I'm gonna talk about also more long-term projects that we are working on to try to again engineer, improve these associations between plants and microbes, to try to take better advantage of the microbes in agriculture. So first, let's start with my yin yang sign on the top which is often the way that people have in mind when they think about symbiosis. They think about something which is mutually beneficial. Well, it's almost right. In fact what we call symbiosis, the definition was coined by Anton de Bary in 1879, as a prolonged living together of different organisms that is beneficial for at least one of them. And that may be the surprise for you because many of you may think it has to be beneficial for both of them. Well, the reason we say at least one of them is that in symbiotic associations we want to include three types of interactions. The one that you probably had in mind, which is the mutualistic association on the left, which is when these relationships are beneficial for both partners. That's what most people think when they think about symbiosis. But in fact, things like commensalism, when the association is beneficial for one and neutral for the other partner, is also part of symbiosis, and even more surprisingly, parasitism, when it's beneficial for one and detrimental for the other one, for the other partner, it's also part of what we call symbiosis. The reason we have such a fairly large definition for symbiosis is that in real life, in nature, every association is going to shift one end from the other. Some associations are mostly mutualistic most of the time, but some are sometimes depending in particular on the environment or let's say even the age of the plants, sometimes these associations are going to turn more to the parasitic end, and you have really nature that continuum. Of course what we want to do as agronomists, is to have associations that are as beneficial for the crop and we try to shift that continuum to where its a more mutualistic association. So, that's what we mean when we talk about symbiosis and so we should not be surprised if sometimes even some of the very classical symbiotic associations that we use, like I'm gonna talk about soy beans and bacterial (mumbles) can be sometimes more into a parasitism. It's just the nature of these relationships. Regarding plants and microbes, so that's really what we are focusing on today, we are talking about these relationships between plants and microbes. What is the deal, what are the exchanges that take place between plants and microbes. So my pine tree symbolizes the plant, and what plans are good at, they are good at doing photosynthesis. They are good at getting carbon from the atmosphere and producing sugar, photosynthesis. And that's really the main nutrient that plants give to their microbes. In addition, they also provide what we call an ecological niche, which is simply a place for the microbes to grow and avoid competition. In return, microbes, and here I used the image of my mushroom, the fungus, is these microbes can really give everything that a plant needs to grow. I don't know if you all have a garden but you know that plants need water, they need nutrients, if you're using a can of fertilizer you have NPK, nitrogen phosphorous potassium. Microbes can provide water, can help plants for the acquisition of water. They can help plants getting their nutrients from the soil or from the air, in same cases, and also these microbes sometimes can help the plants against pathogens, and that is in particular the case of many fungi and bacteria that help protecting their host, their plant, against pathogens. So these are typically the deals, the kind of exchanges that take between plants and microbes. So before continuing, I'm gonna talk about what I call the problem. Why do we really care about studying plant microbe associations. What are the problems that we hope solving or at least alleviating by working with plants and microbes. In my opinion, these problems have to do with agricultural sustainability for developed countries like in the U.S. or in Europe or in China, and also problems of agricultural development in particular for developing countries. That's what I call the growing problem. How can we grow our crops without destroying our environment, or how can we grow our cops to reach the maximum yield possible. So, in developed countries you probably know that we use a lot of fertilizer, and in particular, a lot of nitrogen fertilizer. This nitrogen fertilizer is coming from natural gas. We use a process the Haber-Bosch process that was invented at the beginning of the 20th century, to use the energy coming from natural gas to convert nitrogen from the atmosphere, nitrogen that we call dinitrogen into ammonium. That chemical reaction requires a lot of energy. You need about one ton of natural gas to produce one ton of fertilizer. At a global scale in the world, we use 2% of the world's energy to make fertilizers. So, we spend a lot of energy, we use a lot of fossil fuel to produce that fertilizer and that's a great things because that's what use to grow our crops. On this graph here, I'm sorry I apologize because it's a bit complex, what I want to focus on is that line, that black line that increases, the dark one is the world population. As you know currently, we have about 7 billion people in the world. The other line I want you to look at is that dotted line, black dotted line, that starts about in 1920s and that keeps increasing and that line represents the percentage of the world population that is fed on that Haber-Bosch process, that is fed using chemical fertilizers. As you can see, that started at the beginning of the 20th century, that's when this process was invented, but you have a really drastic increase in the sixties, seventies. That time is what we call the green revolution. One key component of the green revolution in the sixties and seventies was to intensify the use of nitrogen fertilizers. Currently, you see that dotted line, it goes up to about 50%. Currently, half of the world population is fed using nitrogen fertilizers. It's obviously a great thing. Without nitrogen fertilizers, we would not be able to feed half of the world, so obviously nitrogen fertilizers are absolutely necessary. So that's obviously a plus for them. Something which is more worrying is that we make these nitrogen fertilizers out of fossil fuel, out of natural gas, and it's a finite resource and we know that well, sooner or later, we won't have so much fossil fuel available. If you see, we are using fossil fuel to produce fertilizers to grow half of the food of the world, to grow also feed for animal which is something even more ironic, we use these fertilizers to produce bio-fuel, to make bio-energy, so we are using fossil fuel to produce bio-fuel. So obviously I think it makes sense that it's not really sustainable, so I think that's one aspect which is obvious, so, yes we're getting questions, should we ask questions I think at the end, sorry. Great. One of the consequence of that relationship between natural gas and fertilizers is that the price of these nitrogen fertilizers is highly dependent on the price of gas. What I show you on that graph in blue what you have is the price of natural gas. As you can see, there was a peak in 2001 and that corresponds to a peak in the cost of fertilizers in red. And again, when the price of natural gas is increasing, the cost of fertilizers is increasing. I don't show you the graph after that, in fact it's actually decreasing. The price of fertilizers and the price of natural gas are decreasing currently due to fracking but we know that is a finite resource. We know that sooner or later these prices of the natural gas, the price of natural gas is going to increase again and that is going to drive again the price, the cost of fertilizers. So there are economical consequences on that dependence, relationship between natural gas and fertilizers. Another aspect, health consequences. As you probably know, the maximum allowed, maximum concentration of ammonia in drinking water is 10 ppm. It's unsafe to consume water that has more than 10 ppm of nitrate. And what I show you here is the percentage of wells in Rock County, so Wisconsin, that have levels of nitrate above that limit of 10 ppm. When I came here in 2004, it was about 25% of these wells. As you can see, over the years, now we are at about 50% of the wells. So what happens here is that all the fertilizer that we apply for agriculture leaches down into ground water and contaminates that ground water, and contaminates these wells and this is obviously a concern so these high levels of nitrate in drinking water are just directly the consequence of our intensive use of nitrogen fertilizers. Well, that nitrate doesn't end up just in ground water. It's going to go after in rivers, in particular for us here in the Mississippi River, and that has really large-scale ecological consequences. One of them is the formation of what we call dead zones. So what that image represents in green, these are the sites where of intensive use of fertilizers. In pink that represents nitrogen coming from human activity and from cities in particular. But all that nitrogen ends up in ground water, ends up in the Mississippi River and ends up fairly rapidly in the Gulf of Mexico. In the Gulf of Mexico what it's causing is that all that nitrogen, all that nitrate is feeding the growth of algae. These algae consume oxygen and you end up with a region that has very low oxygen levels that basically cannot sustain any other form of life. Fishes in particular cannot grow because of these algae blooms. And that dead zone, which is in the Gulf of Mexico, is a direct consequence of the intensive use of fertilizers in the Midwest. Well, that's not the only place in the world at all where we have dead zones. There are dead zones around Europe, around France. Now, massive dead zones around China, but all that is due to our human activity. That's one consequence, there is also even more global consequence of fertilizers on global warming because one product of degradation of nitrogen to soil is nitrous oxide, it's a greenhouse gas, which is also massive and major component of global warming. So now, all these are ecological consequences of our intensive use of fertilizers. So, on one hand fertilizers are great. They are feeding half of the world. On the other hand, their intensive use has a lot of health, economical and ecological consequences. And that's the kind of problem that we have here in developed countries. In the opposite spectrum, for developing countries, their problem is that they simply cannot afford fertilizers. In fact, in many developing countries the problem is just to get a bag of nitrogen fertilizer to the poor farmers. And what I show you here, before I freak out, in particular, it's a map that shows the places in the world where corn production is limited by nutrient availability. So, everywhere you have a green dot, it means that we can reach 50% or more of the possible yield we can get with the corn. You see in the U.S., in the Midwest or in Europe, no problem. We use a lot of fertilizers. Most of the time we can reach 50 or most this maximum yield. But in places like Africa, in particular, you see where you have red dots or blue dots, these are limited in nutrients and the main nutrient which is limiting in these places, it's nitrogen, the second one would be phosphorous, but the main one is clearly nitrogen. So, for these countries, for these developing countries, their problem is to get that fertilizer and to have access to nitrogen fertilizers. So, nitrogen fertilizers are a great thing but here we have too much of a good thing and over there they cannot actually get it. So, that's the kind of problem that I think we can help solving or at least alleviating with microbes. I'm gonna talk about two types of microbes. Some microbes that help the plants getting nutrients from the soil and getting nitrogen, in particular, from the soil and phosphorous too, and I'm gonna talk about microbes that help the plant getting nitrogen from the air, which is even better. So that's my introductions to these different microbes that I'm going to talk about today. On one hand, we work with microbes that we call mycorrhizal fungi, that is what is on the right part of my figure. These fungi colonize the roots and extend their high feet into the soil and they help the plant getting nutrients and water. They really help pumping these nutrients out of the soil, and help the plant accessing these nutrients. On the left part of my slide, I'm talking about bacteria, bacteria that can do something called nitrogen fixation, which is to convert nitrogen from the air into ammonium, exactly the same process as this Haber-Bosch process that I mentioned before, the chemical process, except that here you don't buy bacteria, or living organisms. Same idea, you take nitrogen from the air and you give it to the plant. So, I'm gonna give you a quick introduction about both types of relationships. These first fungi, the mycorrhizal fungi, when we say mycorrhizal fungi it encompasses a wide range in fact of associations. We classify them into you can see these five different categories of associations between plants and microbes. The ones I'm going to talk about mostly today are the ones on the right called, arbuscular mycorrhizal. And the reason I'm going to focus on that one, and we also focus on that a lot on our research, is that these arbuscular mycorrhizal fungi can colonize 80% of plants and the vast majority of our crops, so these are in agriculture, for us these are by far the major players. You have other called ectomycorrhizal fungi. Maybe you have heard about these ones. These are those that you find in forests. These ones are important, for instance, for poplar production or pine production. You go to any forest, the underground is full of these ectomycorrhizal fungi. Ericoid mycorrhizae, that one is a bit special to Wisconsin, because that's a mycorrhizal fungus that associates with cranberries, so it has a very narrow host. Really, it's about berries. So, ericoid mycorrhizae, honestly not so many people work on ericoid mycorrhizae. A lot of the research is done on the arbuscular mycorrhizae because these ones, as I said, colonize most of our crops. These arbuscular mycorrhizae are very ancient. These are very ancient association between plants and microbes. The characteristic of these arbuscular mycorrhizal associations is what is how you on the right, are these fungal structures that are in blue called arbuscules. And these structures are fungal structures, highly ramified fungal structures that help increase the surface of contact between the plant and the microbes. Now the site where these nutrient exchanges that I'm gonna talk about take place and are very typical of these mycorrhizal associations. When we see such a structure, we know that we have one of these arbuscular mycorrhizal fungi. On the left part of my slide, what I show you is a fossil, a fossil which is 400 billion years old, and 400 billion years ago what happened is that the plant started to colonize land, so this is a fossil of one of the first, some of the first land plants, and inside of these fossils we find arbuscular mycorrhizal fungi. Many of us that study the evolution of these associations think that the ability of plants to associate with these fungi allowed, in fact, plants to colonize land. It's because plants were able to associate with these fungi and use these fungi to extract nutrients from the soil, that they have actually in fact been able to colonize land. So when we think about these arbuscular mycorrhizal association, we often refer to the mother of archebiosis because it's a very ancient association between plants and microbes, and later, I won't have time to talk about that today, but when plants want to associate with other microbes, they often use actually mechanisms that they initially developed to associate with these fungi, so plants have developed initially that association with these mycorrhizal fungi and then used these mechanisms over and over to associate with beneficial microbes. I'm gonna show you a quick movie about what I show you here in white it's one of these mycorrhizal fungi in yellow these are plant cells. As soon as one of these fungi starts touching a plant cell, within just a few minutes you can see that the nucleus of the plant cell is gonna move right under the point of contact with the fungus, and then you see that nucleus moving down and it's followed by a tube, an empty tube in fact, and that empty tube is the path that the fungus is gonna use to penetrate inside of the plant. Once the tube is ready, then the fungus is gonna penetrate inside of the plant cell. That process is really driven by the plant. The plant prepares the path for that fungus to colonize and then it goes through the plant cells, literally through them, it's a tube within the plant cell. Then typically the fungus as you can see goes between the plant cells in the inner part of the root, what we call the cortex, and its within that cortex that these fungi are gonna form what we call these arbuscular structures that I showed you, with a process which is very similar again driven by the plant you see the nucleus starts to enlarge and then the nucleus is gonna move again and create that tube inside of the plant's cell inside of which the fungus is gonna penetrate. So in that process, the path of the fungus is really guided by the plant and it's really mutual communication and mutually some of my colleagues refer to mutual dance between the plant and the fungus, there to establish that kind of association. I think it's fairly intuitive that if you want to have such a high coordination between the plant and the fungus you need to exchange signals all the time. The plant needs to know that that fungus is not a pathogen, that it's totally okay to create a tube inside of a cell to accommodate the fungus. And that's really what we're talking about when we talk about these sweet talks, about how the plant and the microbe communicate because in order to establish such a complex dance, if you want, you really need to have the plant and the fungus communicate all the time and exchange signals in order to recognize each other. So that's my introduction for you to give a feeling of what we talk about, what we're thinking about when we think about signal exchanges. We need the tight coordination between the plant cell and the fungus in order to have these associations. Once these associations are established then you can have exchange of nutrients, in particular through these structures that we call these arbuscules. What I show you here in this figure, I'm not gonna go into the details of all these transporters, but on the right part that represents the soil, the center that's the fungus and on the left, that's the plant. And at the interface between the fungus and the soil, you have these transporters that many of my colleagues are studying that help the fungus getting nutrients out of the soil. You have transporter for phosphorous that have been very well-categorized, transporters for ammonium nitrate, amino acids or urea, which are sources of nitrogen. So really the fungus is pumping all these nutrients from the soil, transporting them to the plant and on the left part of my figure, these are what happen at the interface between the fungus and the plant where again, what's going to happen is the fungus is going to transfer that phosphorous taken from the soil to the plant, nitrate ammonium nitrogen is gonna be transferred and at the bottom of the slide you have the reward, if you want, the plant rewarding the fungus for providing nutrients in the form, in particular, of glucose and fructose sugars. So that's typically the kind of nutrient exchanges that take place, and a lot of us study how these nutrient exchanges are regulated. That's for the association between plants and mycorrhizal fungi. On the other side of my slide I'm talking about associations with bacteria and these nitrogen-fixing bacteria, so the process of nitrogen fixation is the one I mentioned before of transforming, converting dinitrogen in the air into ammonium, and that process is very important ecological process. What I show you here that what we call the nitrogen cycle. In the nitrogen cycle you can see that we have a lot of nitrogen in the air, in fact 80% of the air is composed of nitrogen. That nitrogen unfortunately is not accessible directly to the plant. Plants cannot access directly that dinitrogen in the air. In order to get that nitrogen, it needs to be in the form of ammonium or nitrate, mostly nitrate. I mentioned what is on the right, what I call industrial fixation, which is that process where we use natural gas to convert that dinitrogen into ammonium and then that nitrogen is used as fertilizers, but what I'm gonna talk about now is the process on the left called biological fixation. Biological fixation is when this process is realized by bacteria in particular that can convert dinitrogen into ammonium. This conversion of nitrogen into that biological nitrogen fixation is extremely extremely important in terms of numbers. I'm not gonna talk about what is called atmospheric fixation, which is done by lightning, in fact every time you have a lightning, it converts a little bit of nitrogen in the air into ammonium. Well, as you can see, lightning are responsible for about 10 million tons of nitrogens fixed per year. Biological nitrogen fixation on land is about 90 to 140 million tons. If you go to the bottom of the slide, industrial fixation, the Haber-Bosch process that I mentioned, is about 80 million tons per year. So when you think about scales, it means that we produce almost as much as fertilizer as biological nitrogen fixation on land. So we can think about how that chemical process also is gonna disrupt the global nitrogen cycle. Of course what we would like to do is to shift the balance and to be able to take much more advantage in or agriculture of biological nitrogen fixation to limit our dependence on nitrogen fertilizers. I really don't think that we can totally eliminate nitrogen fertilizers at all, but it's a matter of using less of them and taking more advantage of biological processes. The enzyme in bacteria that performed that conversion of dinitrogen into ammonium is called the nitrogenase, and that enzyme is again gonna take dinitrogen which is N2 here and convert it into ammonium. In order to do that process, that enzymes requires a lot of energy in the form of ATP. You see 16 ATP molecules required for the conversion of one molecule of dinitrogen into two molecules of ammonium. 16 molecules of ATP, it's a lot of energy for a cell. That process of converting dinitrogen into ammonium is very expensive in energy. Whether we do that with bacteria or we do that chemically with natural gas, it requires a lot of energy. So one of the problems, if you want, that we need to solve when we want to improve, take better advantage of these associations is to make sure that these bacteria have an abundant energy source because it's a very energy-intensive process. Another issue that we need to solve in biological nitrogen fixation is the problem of oxygen. This enzyme is highly sensitive to oxygen and so in every system that you have that is efficient, is performing efficient, biological nitrogen fixation you have a way to protect that enzyme against oxygen. One of the most, or if not the most efficient system that have been developed for evolution is the association between plants of the legume family like soy beans, alfalfa, peas, beans and bacteria that we call rhizobia. And these associations lead to the formation of new organs on plant roots that we call nodules. I don't know if you dig soy bean root or an alfalfa root you will often see these small little bumps on the roots that often are pinkish and these are what we call these nodules. These are little, if you want, houses where the plant is gonna house these nitrogen-fixing bacteria and solve the two problems that I mentioned before, solve the problem of energy. These plants are gonna feed the bacteria with sugars inside of these nodules, and the problem of oxygen, they protect these bacteria against oxygen so that they can fix nitrogen. I'm not gonna go through all the details of that cartoon, but that cartoon represents the different steps of the dance in this case between bacteria and plants to lead to that formation of nodules. What that represents, it's a legume root, if you want a soy bean root, where the bacteria are gonna come in contact with the root hairs. The root hairs are gonna curl around the bacteria, enter the bacteria and form these tubes inside of the plant cells to guide again the bacteria deep inside of the plant root. And in parallel, you have the development of these organs that I'm calling the nodules inside which the process is gonna take place. There is a very striking similarity in the process in the colonization process in that association and the one that I mentioned before, the mycorrhizal association. It's actually from an evolutionary point of view, we know that legumes accommodate these bacteria, these rhizobia using processes that initially had been developed to accommodate the fungi, these mycorrhizal fungi. That's the same process, the plant creates a tube inside of the cell to allow, to give a path to these microbes to colonize. So again, for that association, it really requires a tight coordination between the plant and in that care, the bacteria. It requires a lot of signal exchanges. That for the communication part, for the nutrient exchanges, I'm not gonna go into the details of all these metabolize, but the main message here is that the deal if you want is that these bacteria fix nitrogen, they have that enzyme the nitrogenase, and they are gonna export that nitrogen in the form of ammonium, that's the line at the bottom in blue, or in the form of amino acids, these are the transporters in yellow. So the bacteria get nitrogen from the air, make ammonium and give that nitrogen to the plant and the deal, if you want, in return the plant is gonna provide malate as a carbon source. So here these are not sugars, these are diabolic acid like malates, but the deal is the same. The plant is feeding the bacteria with a source or carbon. So that's the kind of nutrient exchanges that take place. So that was my introduction to these associations. On one hand, mycorrhizal associations that you can find in the vast majority of crops, in corn, soy bean, alfalfa, many, tomatoes, potatoes, whatever. Most of the crops are able to associate with these mycorrhizal fungi and on the left, nitrogen fixing bacteria. There it's mostly limited to legumes like soy beans, soy beans, peas, beans. That's why in agriculture we rely so much on legumes in any sustainable system in agriculture we always need to introduce a legume and the reason why we always want a legume, particularly here in the Midwest, is the corn soybean rotation. The reason we want soy bean is because of that process of biological nitrogen fixation. It's because these soy beans can get their nitrogen directly from the air without the need for input, without the need for fertilizers. So that's the introduction. So I'm gonna talk now about I would say more the core research of what we are doing in my lab as well as in other colleagues are working on too, about what I call the sweet talks and trade deals, which is how plants and microbes can talk to each other, how they can recognize each other, how they can do that little dance of associating together and how they can regulate these exchanges of nutrients. Another way to think about the sweet talks, if you want, is how do you recognize your friends. How plants can differentiate beneficial microbes with other microbes of fungi or these rhizobia from pathogens. How the plant, why the plant is gonna create these tubes inside of the plant's cell for the beneficial microbe and not for pathogen. So what we're working on there are signal exchanges and typically what has been found over the last years and decades of work is that on the left part, for instance, of my talk between plant and mycorrhizal fungi we know now that the signal from the plant to the microbe, the main signals are called strigolactones. They are in fact plant hormones that plants release through their root exudate in the soil. And these signals are gonna trigger the production of factors that we call myc factors or mycorrhizal factors that then the plant is gonna recognize. So it's really a communication system. The plant is producing a signal saying I'm there, really what strigolactones mean is I'm there. The plants say, I am looking for hosts, in fact strigolactone is increased when plants need mycorrhizal associations. And in return, when you have a fungus present, the fungus is gonna produce these myc factors to say, hey, open the door. On the right part of my slide, it's a very similar dialogue. It's again, it's a molecular dialogue where there are plants and microbes talk with molecules. There, the legumes produce flavonoids, isoflavonoids, compounds which are going to be recognized by these rhizobia bacteria. And these bacteria, in return again, are gonna produce signals that we called nod factors for nodulation factors. So when the signals are coming from the microbes we call them mycorrhization factors or nod factors, nodulation factors. What is interesting for us in that dialogue, in that signal exchange, is that nod factors and myc factors are in fact very similar. Nod factors are molecules that we call lipo-chitooligosaccharide. Of course I don't want you to remember the structure, we call them LCOs. They are, if you want, a keratin backbone and a lipid. Interestingly for us, just a few years ago it was found that the signals form the fungi are again what we call myc factors, are again lipo-chitooligosaccharides, these LCO molecules. Not exactly the same structure, but very similar molecules. So what that means on the global scale is that for from the plant perspective, whether your friend is a fungus, is a mycorrhizal fungus or the friend is these rhizobia bacteria that take nitrogen, the signal they perceive are very similar. What these LCO signals mean to the plant is I'm a friend, which is a great thing. It's great for us because now we know what kind of signals microbes use to say, I'm a beneficial microbe. So if you think about it, on one hand it's great because now we have each one the golden key to allow microbes to associate efficiently with crops, and so you think, oh great, we can help increase, improve association between crops and these beneficial microbes. That's true. On the other hand, that can be scary because you think hey, maybe some microbes are gonna find a trick, some microbes are gonna use these LCO molecules to trick the plant and try to be a pathogen. So imagine that you have a microbe which is a pathogen that produces that kind of signal, they have the golden key to colonize a plant. They can trick the plant and tell the plant they are a beneficial microbe when they are not and that's really where my second part of the think we are interested called the trade deals are important. Cause these trade deals, their goal is to deal with cheaters, to deal with microbes that would trick the plant, that would say I'm a beneficial microbe, but without providing any benefit. Imagine that one of these fungus or bacteria can penetrate inside of a plant and do nothing except grow. That's not what you want. That's not what the plant wants. The plant needs to have a system to deal with the cheaters, and these are actually very interesting stories, very briefly that I'm gonna talk about. When I was talking about the signal exchanges, the striking feature is that the signal is pretty much the same whether it's coming from these fungi or the bacteria. They are these LCO signals. When it comes to dealing with cheaters the strategies that plants employ are very different. First, in associations in between legumes and rhizobia, what the legumes are doing, like soy beans, or alfalfa is doing, is what we call a sanction mechanism. It means that the plant is gonna kill the nitrogen fixing bacteria that are not efficient. If you have some of these bacteria that produce the LCOs but do not fix nitrogen efficiently, they are gonna be killed by the plant and these are very interesting experiments that were performed by a colleague in Minnesota where what you see on the left are, I don't know if you can see, but you have a root with small nodules and on top you have these fancy equipment that is used to control the atmosphere around the nodules. And what they are doing here is that they are tricking, they are using a trick to prevent these nitrogen fixing bacteria from fixing nitrogen by replacing the atmosphere around the nodules by argon. So in the air what you have it's in the right in the air in black, normal air has nitrogen and oxygen. If you replace the nitrogen by argon, the bacteria cannot fix nitrogen anymore and that's a trick that we use to prevent some nodules from fixing nitrogen whereas we can allow the nodule next to it to still fix nitrogen. What happens when you replace nitrogen so the black with argon, the gray one, is that what you see on that graph is a decrease of the number of rhizobia inside of these nodules. In other words, when you prevent these nodules from fixing nitrogen if for any reason one of these nodule structure on the soybean roots don't fix nitrogen, then the plant is gonna kill the bacteria inside of it. And that's a sanction mechanism, and it's very efficient, you can imagine, at selecting cheaters. Only the bacteria that can fix nitrogen properly inside of this structure will be able to grow, multiply and later be released into the soil, whereas the cheaters, of course they can go into these modules but they are going to be killed by the plant. So, in that plant bacteria association, it's a sanction mechanism. Totally different from what we see in mycorrhizal associations and that is I think beautiful. There, that mutualistic association is stabilized by rewards system, in fact it's a mutual reward system. The plant is gonna reward the good fungus and the good fungi are gonna reward the good plant, and what I show you here I experiment so the top part of that experiment were performed by a colleague, a fantastic colleague, Toby Kiers. What she did is that on top you have a system with three compartments. You have a root compartment and then two fungal compartments on the right and on the left. On the right fungal compartment, these fungal hyphae have access to phosphorous, they can get phosphorous. On the left, they cannot get phosphorous. So what happens here in that system is that both fungi those that can access phosphorous and those that cannot can colonize the same plant. And what she's doing is that she's monitoring how much sugar that plant can give to the fungus by using (mumbles). And what you have on the right, what this shows is the amount of sugar that the plant gives to different fungi. On the fungi that cannot provide phosphorous or have zero micromolar, you see far less sugar given to that fungus than to the fungus that can have access to have nitrogen whether it's 35 micromolar or 700 micromolar of phosphorous. What that means here, is that when the plant has the choice between the fungus providing phosphorous and the fungus not providing phosphorous, the plant is gonna give its carbon to the fungus providing phosphorous. So the plant is gonna reward the fungus giving it benefit. Reciprocal experiment that the top bottom part of my slide, again, that's the same design. Three compartments except here it's one fungus and two plants, two root systems, one root system on the right that can provide sugar and one root system on the left that cannot, and what she measured here is how much phosphorous is given by a specific fungus to two different root system, a root system that can give sugar and a root system that cannot give sugar. What she observed that's what is on the right is that the fungus is gonna give it's phosphorous to the root system that can provide the sugar. So in other words, this fungi when they have access and that's the case in nature, these fungi can colonize many plants at the same time, these fungi are gonna give their phosphorous to the plant providing the more carbon. It's a reward system, and so you have that mutual reward system where the plant rewards the fungus that is providing the most benefits and the fungus rewards the plant that provides the most carbon. So it's that mutual reward system that stabilizes that association and makes sure that you avoid cheaters, because only the most performing partner is gonna be rewarded. So that's really what we study when we study these trade deals, and how we deal with cheaters. So that's pretty much what we are doing in my lab, working on these signal exchanges that the sweet talk, how they can recognize their friends, and on the right, how they deal with cheaters. Briefly, with the time I have left, I'm gonna talk about applications. Some of these applications are what I call the low-hanging fruit, that are the practical applications of how we use these microbes in the field. These microbes, many of these microbes are used and commercialized as inoculants If you buy beans or peas for your garden, you often find next to the seeds, you can finds bags of rhizobia which is sod, and these rhizobia are applied typically on the seeds before planting. Soy bean farmers know very well that they need to inoculate fairly frequently with these rhizobia. In fact, I have a piece of the Wisconsin State Journal from 1906 recommending to farmers to inoculate their seeds. The University of Wisconsin has been one of the first ones providing their farmers with these rhizobia inoculants. At the time, in the beginning of the 20th century, it was top technology. Now it's widely used. What we're currently doing with that kind of technology, is try to evaluate when these products, whether they are rhizobia inoculants or mycorrhizal inoculants, when they're really useful for the farmer. When do they really need them, when they don't need them. Mycorrhizal inoculants, in particular, are pretty expensive. The rhizobia one, they are pretty cheap, but in both cases what we are doing is try to inform the farmer about when these products are useful, when they are necessary, what are the best products on the market. So that's I would say a very direct application of the work. A bit more development side, I mentioned that these NaCL-molecules that are these key signals produced by these rhizobia or these mycorrhizal fungi. In fact, for about 10 years now, almost 10 years, these NaCL molecules have been commercialized as plant growth promoters. In fact, what these signals mean to the plant, is that you have beneficial microbes around, and one direct consequence of that on the plant, is that the plant is gonna grow faster. The plant thinks that you have beneficial microbe it's gonna develop, in particular, its root system to be colonized, so we treat the plant but that has a direct consequence, in particular, that really helps for establishment, for crop establishment. Sometimes the crop is gonna grow a bit slowly at the beginning of the season. If we treat with these products, the crop is gonna grow much faster at the beginning. And that directly translates into here. So what we are doing is working on one hand, we are finding more of these structures of this NaCL molecules and we find things that work better, and on the other hand, we also work on efficiency, on when these products are useful for farmers and when they are not so useful, and so we try to give some guidance of when they should be used and when that's not necessary. So all these are direct consequences of the work we do on these bacteria, on these bacteria rhizobia, on these fungi because we have commercial products available we try to inform the farmers and possibly develop new products. More long term application of what we are doing is to study evolution and to study how these associations evolved and in particular things that we're really excited about is to understand what are the critical innovations, what happened during plant evolution that allowed plants to form these arbuscular mycorrhizal associations 150 million years ago. So what I show you here, it's a very simplified tree, phylogenetic tree of land plants and at the base of that tree you have these arbuscular mycorrhizal associations because as I told you, plants as soon as they started colonize land, they started forming these associations, so the kind of research we do is, for instance, comparing algae to some of these most basal land plant called liver warts, and figuring out what happened, algae do not form mycorrhizal associations, liver warts do. What happened at that point of time that allowed plants to form these associations. And eventually later, during plant evolution what happens in legumes. Why legumes got the ability to associate with these rhizobia, whereas for instance, corn or cereals cannot, and the reason we are so interested in evolution is that these evolutionary information can guide our engineering projects, and that's really why we study, evolution is interesting in itself of course, but also it has some practical applications. We can use that information to engineer, to create new things. Legumes are going to associate with rhizobia. Cereals like corn, wheat, rice cannot associate with rhizobia. Can we transfer that ability to associate with nitrogen fixing bacteria from legumes to cereals, in particular, and that would be fantastic because that would help reducing our dependence on nitrogen fertilizers. And so that's my, I think it's a pretty cool slide. It's my yes we can. That's the slide I was using to try to convince the community that yes we can engineer symbiotic associations, and that's really one of our major goals here is to try to improve the associations between cereals, in particular, and nitrogen fixing bacteria. In agriculture, growing corn is one of the major reasons why we use so much fertilizer. Cereals require a lot of nitrogen fertilizer and if we could at least alleviate the dependence, reduce the dependence on nitrogen fertilizers then we would really have a big impact. So that's the kind of project that we're really excited about. It's funded both by the Bill and Melinda Gates Foundation and the reason they are really funding that kind of project is in particular for Africa and for poor farmers. I was mentioning at the beginning the problem. First, the problem here is that we use too much fertilizer. For them in Africa, the problem is access to fertilizer, so the Gates Foundation is funding that kind of project in particular to alleviate the need of fertilizers in Africa. And the NSF, National Science Foundation, is also funding that kind of project. Of course it's really long-term but I think for us it's really the Holy Grail. That's really what we want to achieve in the long term is to allow cereals to grow with less fertilizers, and that's really where we think we can have an impact on agriculture. Alright so that's the overall view of what I talked about. I just want to take a few seconds to acknowledge my lab members, the people doing the work; Funding is coming from NSF, the DOE, and the USDA, and the Gates Foundation; and all these great students that I have the chance to work with, as well as colleagues who are right here on campus from very different discipline, from biochemistry to microbiology and plant sciences. It's a really interdisciplinary work. We work with plants, we work with fungi, we work with bacteria and we take that from different angles, and one of my students is here actually, in fact, in the room. Alright? So, thank you very much for your attention. It was pleasure to be here. (applause)