Wisconsin Public Television University Place Wednesday Nite at the Lab  Laura Helft When Microbes Attack, Plants Fight Back  August 24 2011 UP614     cc >> Laura Helft: Good evening, everyone. My name is Laura Helft, I'm a graduate student here at the University of Wisconsin-Madison. The new students just got here a couple days ago for our program, and so that officially makes me a six-year graduate student, which puts me in the 22nd grade is what I figure. (laughter) So hopefully, I have a lot to tell you that's pretty interesting tonight, because I've been at this school thing for a while. So I'm a PhD candidate in the Cellular and Molecular Biology program. I'm going to be describing the work that I've been working on in the laboratory of Andrew Bent, which is in the Department of Plant Pathology, talking about how plants are actually able to recognize the microbes that are around them, which isn't maybe something that you think about often. I certainly didn't think about it very often at all before I came to graduate school. But now I find myself thinking about it a lot. Not just because I have to, but because I think it's very interesting. So, to give you a little bit of background, I just want to quickly start by giving everyone here sort of a whirlwind tour of plant pathology, so how we know plants are sick, what we know about plant diseases, what we know about the plant immune system, what we know about how microbes actually are able to attack plants. 

Why we care about this in the first place. What sorts of things microbes have in their arsenal to actually make plants sick. Then I'm going to introduce the plant that we actually study: Arabidopsis, which you might have never heard of before. Maybe you're thinking of arabi-whatsis? But hopefully by the end of this talk, you'll have a better idea of what that plant is. Then finally, talking about the plant immune system. What does the plant immune system do, and how does that actually function? How are plants actually able to sense the presence of bacteria? So that'll be the first half. Then I'll be talking a bit about the research that I'm actually doing in the lab, which is basically helping plants sense pathogens better. So while some plants are very good, it seems that when there are things around that can make them sick, they're not always the best at it. So one of the things I'm trying to do with my research is make plants better at recognizing the different things that can make them sick. So, why do we care about this in the first place? So what if a few plants get sick? It's not a big deal, there's billions of them on the face of the earth, right? Well, as it turns out, there's also billions of us on the face of the earth, and this is just a graph showing, which some of you may have seen before, of human population throughout history. 

So you can see there really haven't been many of us around for very long. You can see there's-- actually right here, it's pretty cool, you can actually see a dip in the population where the Plague actually occurred. But since then, growth has been increasing steadily, steadily, steadily. We're now up to about 7 billion people in the world. So what does that have to do with plants? Well, plants are at the bottom of the food chain, so, if you don't have as many plants around because they're getting sick, you have a problem for feeding all these people. I think it's a bit hard to see on this screen, but what there is here is this red kind of faint line is actually the population, the human population in the world. Then this yellow, whitish line is the food supply. And what you can see here is the food supply line is actually sort of starting to taper off a bit. That's a problem. If we need to feed all these people that are present in the world, we need to have food. 

It's estimated that about 10% of all crop loss worldwide is due to pathogens. So, pathogens eating the plants, making them sick, making them unable to produce as much for the people growing those crops. So as plant pathologists, were very interested in, how does this interaction between microbes and plants actually happen? So what does a microbe actually look like when it's attacking a plant? I bet a lot of you have seen these and you just had no idea that there was a microbe involved. A lot of times, it looks very similar to something you might see when a plant doesn't get enough water, when it gets too much sun. The same sorts of symptoms will often arise. So here on the left is a picture of a plant. Does anyone have any inkling as to what this plant is? Any? No? It's grown here in Wisconsin, and actually you eat the portion of the plant that's underground, which is why you might not recognize it. It's a potato. This is what a potato plant looks like growing out in the field. So this is you know, very happy, healthy potato plant, but if you get a disease called Phytophthora infestans, within about 72 hours, you can go from looking like the plant on the left to looking like the plant on the right. 

So you can see that the plant has a whole bunch of these spots all over its leaves. Basically, there's a fungus that's attacking the plant, and liquefying it's tissues, essentially, which is pretty gross for the plant. If you destroy the tissue, you're going to destroy the crop. You might have heard of potato late blight before, because this is actually the cause of the Irish Potato Famine. So about 150 years ago, there were many people who immigrated from Ireland into the United States and a major cause of that immigration was due to the fact that conditions were very favorable two years in a row for the growth of this pathogen basically wiping out the the largest crop in Ireland and causing many people to need to immigrate. You may have also heard of late blight in the context of Wisconsin. Potentially, if we have some gardeners in the audience who grow tomatoes, maybe some of you are weeping when you see this picture, because it looks eerily similar to something that you saw two years ago in your own garden. This is a picture of late blight on tomato. You can see these plants in the front are just starting to succumb, and then if you go further back, you can see the plants looking worse and worse, something like a tomato plant you might see later in the season, towards September or early October. But this is, again, caused by a fungus that's very, very similar to the fungus that causes potato late blight. 

So it's not just that plant pathogens were a problem back 150 years ago. They're still a problem today. Potentially another plant pathogen that you may have seen, we don't see it as much anymore, because most of the hosts have been destroyed is Dutch Elm Disease. I see a few people nodding in the audience and reaching for their Kleenex, because this is one of those diseases that I find makes people very emotive, because everyone sort of has, remembers elms trees that were around maybe 30, 40 years ago, and since have succumbed to Dutch Elm Disease. So you can see, here's a picture of a tree, and you can see that the fungus here is really starting to go to town. I'm sure that since this photo's been taken, that the tree has had to have been removed. Dutch Elm was a disease that scientists are fairly certain at this point, came over from Asia in logging materials that were brought over about 40, 40, 50 years ago. Because the trees in this country had never seen the disease before, they were completely susceptible, and pretty much we are almost completely out of elm trees at this point. It's not just fungus. I've talked about a few fungi that cause diseases. 

It's not just fungi that cause plant diseases. Bacteria can also cause plant disease. So, one of the bacteria that we're actually interested in studying in my research is a bacteria called Erwnia amylovora, and it causes what's called fire blight on apple. The reason it's called fire blight, is what you can see the leaves look like they've, on these plants, have been completely scorched. They're just brown and really, really horrible looking. So, this can infect apples. It can also infect other related species, like pear. But one of the big problems with this, like any disease that attacks a tree, is, as you know, a tree doesn't grow overnight. So, if you are an apple farmer, and if you get this disease in your orchard, you basically have to start over from scratch, which for an apple farmer, can take ten to 15 years. This is a big, big problem among apple growers, and people who have apple trees are very careful to make sure that they spray religiously to keep this, or this bacteria off their plant. 

In addition to bacteria and fungi that can cause plant diseases, there are also nematodes that cause plant disease. Nematodes are small worms. They're less than a millimeter in length, that are able to also cause plant disease. Here, what I'm showing you is a picture of a soybean root. Here's the root, but then they're all, you can see these sort of round shapes coming off of the root. This is caused by what's known as a soybean cyst nematode. These round structures are actually cysts. So what these are, is actually a nematode that's gone into a plant, infected the plant, and then produced eggs, and then the body of the nematode actually hardens around the eggs, and sort of produces this like casing to protect the eggs before the body ruptures and then the eggs are released back into the soil, starting the disease cycle again. So basically what these pathogens do, is they divert all the resources from, instead of going into making more soybeans, they divert the resources into making more nematodes. So, as a result, this can have a severe impact for farmers in terms of how many soybeans they're actually able to produce from a field. 

We have fungi. We have bacteria. We have nematodes. All of them can cause disease. We also have viruses. This is probably a plant you've never seen in Wisconsin. This is a picture of a papaya leaf, which is one of the major cash crops in Hawaii, as well as other tropical islands. About 25 years ago, they had a really big problem with the papaya ring spot virus. You can see that the leaf on the right, it's a little unclear in here, but it's, the leaves are somewhat mottled looking so they sort of start to get yellowy. If you look closer, they actually form sort of a ring like appearance, and so that's why it's called ring spot. 

What this is actually caused by is a virus going in and making the plants sick. So, just like we can be infected with fungi, or bacteria, or viruses, plants also can succumb to all these different types of pathogens.  So, to me one of the things that I think is also really interesting about plant pathogens is that people have also become more interested in them post 9/11, because there's some more concern that they could become agents of bio terrorism. So after 9/11, there's some legislation passed that basically listed plant pathogens as what are called select agents. So you have to be highly trained and certified to actually handle these, just like if you were to handle anthrax or Ebola. The reason that they gave them so much-- that they spent so much time thinking it would be as important is because if these pathogens were to become epidemic in this country, spread across the country and actually infect many of our crops, we would have a serious food shortage on our hands. I think it's interesting that, you know, we often like to think a lot about plant diseases, or human diseases, and the impact they could have on us, because it's very immediate, but if you impact our food chain, we're going to be in pretty much just as much trouble as if we were attacked by a human pathogen. So, I just spent the last ten, 15 minutes or so giving microbes a pretty bad rap, but I want to make it clear that just like not all microbes are bad for us, not all microbes are bad for plants either. So, to just illustrate very briefly that microbes are important for plants as well, here I'm showing you another picture of a root. This is actually a root from an alfalfa plant and again, you see these sort of cysts or sort of nodule-like structures on the plant and you might think, it's another plant pathogen causing disease. 

But in fact these are structures that a plant would like to have around. These are structures called nodules. They are filled with bacteria that, their job is to make nitrogen available to the plant. So, these are actually bacteria that the plant need around. Just like we have healthy bacteria that live in our gut and help us digest our food, plants have healthy bacteria around that help them fix nitrogen and make it available to them, so they can grow and thrive.  So, how do microbes spread? Much like microbes spread for human disease, plant pathogens can spread in similar ways. So, globally, as I mentioned before in the example of Dutch Elm Disease, you have movement of new diseases into previously uninfected regions. So, just like with a human pathogen, if you introduce a microbe that's able to cause disease in plants into a place that the plants have never seen this microbe before, you're going to end up with a lot of very sick plants. To spread things from region to region, one of the biggest carriers of plant diseases are insects. This is particularly true when it comes to viruses. 

So oftentimes, viruses, which are extremely small, are able to actually attach selectively onto the mouth parts of an insect as it's feeding on one plant that's already infected with the virus. Then as the insect moves to another uninfected plant, the virus comes off, attaches to that plant, and then infects that plant and makes it sick. So insects are actually a major, major source of microbe spread from plant to plant. Then locally, you can also get microbe spread through splashing, so through rain splash. So, you could imagine sort of like if you had an open wound, plants can also get open wounds that are filled with nasty bacteria and fungi, and if you imagine having one of these wounds sitting out in the field and it rains, the drops from that rain hitting that will actually spread the bacteria to neighboring plants. That's oftentimes how infections can spread locally from plant to plant. In the case of fungi, you can also get what's called sporulation, so if you've ever seen one of those puff ball mushrooms that you might find in a field. You can pick it up and squish it and will sort of release this dust like stuff into the air, that's actually the spores, so that's the reproductive structure for that microbe. Fungi that infect plants also are able to make spores in order to spread themselves out locally. I've talked a bit about the different types of plant pathogens we have, different ways that they spread, different ways that they make plants sick. 

As I mentioned earlier, I'm interested in this bacteria that causes fire blight on apple trees. The problem with apple trees is they're not very easy to grow in a lab. Our lab is approximately half the size of this room. We can't fit very many apple trees in there. And as I mentioned, I'm a sixth-year graduate student. If I were trying to grow my own apple trees to study, I would probably be a 26-year graduate student before I could graduate. Oftentimes in the lab, scientists will actually work with what are called model plants. The model plant that I work with is called Arabidopsis thaliana. We usually just refer to it as Arabidopsis. So, you've probably seen its relatives actually growing. 

It grows natively around here. Actually, if you want to, after the talk, usually you can see some sort of in the low lying shrubbery outside the building. I can go point some out to you. But they're fairly small. Once they're full grown, they're about 2-3" in diameter. On the left here, is just a picture of an Arabidopsis plant growing in soil, so you're looking at it from the top down. On the right, this is just a side view, so you can see what it looks like. I would say this is about a four- or five-week-old plant. On the right, you can see that it's now sent up this stalk. This is the stalk it actually sends up when it's switching into a reproductive phase of growth. 

So, why would we study this bizarre little weed that grows around here in Wisconsin? One of the reasons that I mentioned before, it's small. Although our lab is only about half the size of this room, we have about 10,000 of these plants growing right now in our lab, and it doesn't take up very much space at all. They're also self-fertile, which is really convenient, because if they were not self-fertile, it means that every time we wanted to get a new generation of plants, we would have to cross them, which is a very time consuming process. But this way, the plants do all the work for us. We don't have to worry about anything. They'll produce seeds all on their own. They also have a short generation time, so as I mentioned, the plants I was showing you before were about four weeks old. In about two more weeks, the plants I showed you would have set seed, and you can harvest that seed. So you can go from seed to seed in about six weeks, which is great for doing experiments. And in addition to producing seed quickly, they also produce a lot of seed, so you can easily get thousands of seed from any given plant, which is really great for doing lots of interesting genetic studies with these. 

Conveniently, the genome is also sequenced, so we know a lot of information about the genes that this organism has and what they do. There's also a large genetic knockout collection available. What I mean by that, is that because there's so many researchers that work on this plant, there's a stock center that basically keeps lines of these plants that are, each line is missing a different gene in the genome, and it basically covers the entire genome. So, if we want to see the effects of missing a particular gene on Arabidopsis, we call it the stock center. Five dollars later, and a week's worth of time, we have the seed that is a knockout for that particular gene, lacks that gene, which is a really, really nice resource to have. It's also, in addition to having a large collection of plants that are missing genes, it's also very easy to transform these genetically, which means put new DNA into them, put new genes into the plants. So, these are great plants, because they're very genetically manipulable. They're great to do lots of experiments with genetically. They're very amenable to moving DNA in and out of, which is great. So, I'm giving you a little bit of background about how bacteria actually cause disease, and what that actually looks like, but I wanted to give you a little bit more background, molecularly, of what does it actually look like for the bacteria? What do they actually have that can make plants sick?  So here's our slightly simplified bacteria cell. 

One of the most important things that a bacteria cell has is a shield surrounding it to keep anything out that it doesn't want to get in into the cell. So it's basically like having a shield. This is a structure that's called a cell wall, which surrounds the entire cell. Now, it's great to be able to be transmitted by an insect or by a rain splash, but it's also nice to get where you want to go yourself. So, they also, bacteria also often will have a tail on the back, which allows them to move around and this tail is called a flagella. You'll want to remember this for later, because this is actually part of what I study, is the flagella from this fire blight pathogen and how plants are actually able to recognize that tail. So, we have the bacteria protected, we have it able to move around, but of course we need some stuff to go in and cause some collateral damage. So bacteria are also full of all kinds of molecules that are able to make a plant more hospitable to them, so be that changing the pH of cells, to forcing cells to basically release the sugars that they're trying to keep for themselves and give it up to the microbe. There are thousands of different molecules that bacteria have that are important for causing disease in plants. So, you might be wondering, so we have all these molecules inside the bacteria, well, how do they actually get into the plant? Bacteria actually have this pretty cool system. 

It's actually sort of like a syringe that a bacteria is able to produce. What it does is it actually takes these molecules, and it actually injects them into the plant through this syringe-like structure that it has and releases them into the cytoplasm, so that's the inside of the plant cell. Once they're in there, they can go in and cause havoc according to the plant, or according to the microbe, go in and make things far more comfortable for them. A plant is not just going to idly sit by and let this happen, which perhaps some of you may have thought they would. You know, plants don't really move very much per se, but they do have a lot of ways that they respond to things in their environment, including the threat of becoming sick. So here is our slightly simplified plant cell. Plants also have a whole bunch of pieces in their arsenal to allow them to fight back against these microbes. So, tit for tat, just like bacteria have a cell wall surrounding them, plants also have a cell wall that keeps things out that they don't want to get in. And actually, when a plant senses that a microbe is present it will actually make the cell wall thicker, which makes it even more difficult for a bacteria cell to get inside the plant.  And, much like bacteria have molecules that can make plants sick, plants have molecules that can make bacteria sick. 

So, these are basically antibiotics that plants can produce that are actually toxic to the bacteria, but not to plants themselves. And very importantly, plants also have a series of receptors on the surface of their cells that allow them to sense the presence of a microbe. So, just like in our bodies, we have an immune system that goes around and does surveillance for us, basically, looks in our body to see if there's anything foreign that we need to get rid of, plants also have to be constantly surveying to make sure there aren't microbes they need to attack. The difference with plants is that unlike with us, there aren't any specialized plant cells that are just immune cells. Every cell in a plant has to function as an immune cell itself, and be responsible for recognizing the presence of a microbe and destroying that microbe. So if all else fails, plants also have a secret weapon, which is called programmed cell death. It's also know as the hypersensitive response. What this actually does is, if you can imagine, you have an infection, an outbreak that's spiraling out of control, one good way to actually curb that is to kill off all the cells that are infected, and not allow the bacteria to spread any further. It's much like you might have heard of the way that they sometimes will do fire damage control, where they will actually select and burn a ring of trees around the fire so that once the fire reaches the periphery of that ring, it can't spread any further, because there's no other place for the fire to go. Similarly, a plant, when infected with bacteria, if it senses that basically the infection is out of control, it will kill off selectively, the cells that are infected with those bacteria to allow the other cells of the plant to live. 

Plant cells are very noble, clearly. So as I mentioned before, it's really important for plants to know when there are microbes around, because you can imagine you can easily get to a setting where there are bacteria in the cell, in the plant, and they're dividing, out of control, they're taking over the plant, they're using up all the resources, and so the really important thing for plants to have is receptors. Receptors are the pieces that are responsible for actually recognizing the presence of a bacteria. There are basically two classes of receptors that I'm going to talk about just very briefly. One is internal receptors, which recognize these effectors that bacteria are actually injecting into the plant cell.  The second type of receptor, which is the type of receptor I study, are the external receptors that actually lie outside the cells. So you can see, here's our cell membrane, and the receptors here are actually on the surface of the cell. So they can recognize a pathogen before it's even able to put anything inside the cell. So these are basically the first responders that are just sensing to see if a bacteria is at the cell surface of a plant. These receptors can recognize a lot of different molecules, from different types of pathogens, from different types of microbes, and I just want to give you a brief, sort of survey of the types of molecules that they can recognize. 

From viruses, they'll often recognize double stranded RNA, which is basically the DNA of the virus. From fungi, they can recognize the cell walls that they have, which are primarily built of chitin. From bacteria, which is what I study is an interaction between bacteria and plants, they can recognize pieces of their cell wall, pieces of their cell membrane, housekeeping proteins by which I mean proteins that simply are around all the time and keep the bacteria cell alive and happy. In the case of what I'm going to be talking about tonight, they also recognize these tails on the back of bacteria called flagellin. The receptor that recognizes flagellin is a receptor called FLS2. It's called FLS2 just because it recognizes flagellin, so it actually stands for flagellin like sensing two. So FLS2. So this is a receptor that sits on the surface of the cell and this part here is kind of hard to see in the cartoon I've drawn for you here, but the external part sort of looks like a Slinky in mid-tumble down the stairs. It's sort of a spiral shape, but curved over slightly. It basically forms this nice little pocket where a flagellin could come in and actually bind to the molecule. 

Once this receptor recognizes the flagellin, the bacteria tail, it's able to tell the plant cell to launch a number of different responses. I'm not going to go into these in great detail tonight, but the point is that one molecule from a bacteria can actually elicit a wide range of different immune functions that all together lead to the plant's defense against this pathogen and ultimately, in most cases, immunity towards infection by this bacteria. What I'm really interested in, we have this receptor here that's able to recognize flagellin, but how does this recognition actually happen? Furthermore, can we actually change this receptor so that it's able to see more things from a pathogen? Because it's all well and good to be able to see flagellin from some bacteria, but as I'll discuss in just a moment, it's not, these receptors aren't always able to see all flagella from all bacteria. So, why do we care about recognizing flagellin? We know that plants that are able to sense flagellin are more resistant to bacteria. So if you have a plant that's actually able to sense the presence of bacteria by sensing its flagellin, it will have less bacterial growth within it. What I'm showing you here are our friends again, Arabidopsis, that we talked about earlier, the two on the bottom have not been infected with bacteria and the two on the top have been. What I want you to focus on is the difference between this top left plant and this top right plant. Both of them have been infected with bacteria, but the plant on the left is a wild type plant, so it's just like any plant you would find outside. This plant on the right is a special genetic knockout that does not have that FLS2 receptor in it. What you can see is this plant, it's a little difficult to see, if you can't see it, take my word for it. 

This plant is, it's more yellow, it's basically, it has more disease. If you were to go in and actually count the number of bacteria that was inside this plant, you would see there's actually more bacteria growing in here, all because the plant is missing a single receptor, all because this plant cannot sense the presence of flagella, the bacteria tail. So, what we're really interested in, is can we help plants do a better job of recognizing flagellin? Because obviously it is important to immunity.  So, it's one thing to look at plants that are diseased, and we can go in and count bacteria. But how could we know in a cleaner way whether or not a plant has actually seen a piece of bacteria? So I mentioned earlier that there's a whole bunch of responses that FLS2 elicits when it actually binds to flagellin. One of the ones I haven't mentioned yet, but is one we use often in our lab is what's called seedling growth inhibition. All this means is that in the presence of a piece of bacteria that a plant is able to recognize, a plant basically diverts all its resources into turning on defenses and doesn't put any resources into growing. So as a result, if you grow plants in the presence of a piece of bacteria that it's sensing, the plants will be stunted in growth. So here on the left, this an Arabidopsis seedling after about, I would say, a week and a half to two weeks of growing, so this is the whole root down here and everything. But what you can see is this plant on the left is much larger than this plant on the right. 

This plant on the right is the same age but it has been grown in the presence of flagellin. This is an assay we use often in the lab where we'll grow plants in the presence of a molecule that we want to know whether or not it's able to recognize. What we do is we grow them for about ten to 14 days and then we'll weigh them. We'll compare the weight of the plants that have been exposed to that piece of microbe versus the ones that have not been exposed. What we expect, what we're looking for are plants that are actually smaller in the presence of this piece of microbe. So this is basically our read out for whether or not we have a functional FLS2 receptor. So, why did I spend all this time talking about this? We're interested in the flagellin receptor because the bacteria tails from all plant pathogenic bacteria are not equal. Some bacteria species have actually evolved so that their flagellin is no longer able to be recognized by a plant. What I'm showing you here is some data from our lab from about five years ago, where we've grown a whole bunch of different Arabidopsis plants with flagella from different bacteria strains. It's not important that you know what these are, but what I'm showing you here is that there are different responses to different flagellin peptides. 

So over here on our left we have our mock treated plants. What you can see is they grow to be about seven milligrams in about a week and a half, whereas if you expose them to this flagellin, which we know plants recognize, the plants are much, much smaller. They're about one milligram, so that's quite an obvious difference in growth. But when you add flagellin from a number of different species, what you can see is that there's a range of responses that a plant is able to come up with from basically, as good as this sort of what we call, a canonical flagellin peptide, so with a nice reduction in growth, to some of these strains over here, which these three basically, the plants are the same size as a plant that wasn't grown with flagellin. What that tells us is that these plants aren't able to recognize this particular flagellin peptide. So this is interesting because we want to create plants that are able to recognize flagellin better, recognize flagellin from more plant pathogenic species. So what my research has been focusing on, is basically getting these bars here on the left to shrink. So, can we actually get a plant to actually recognize some of these flagellin peptides from other plant pathogenic bacteria. So just to quickly summarize what I've told you so far, plants get sick because they can't see or sense all pathogens. One of the goals that we have is to actually make them able to sense the pathogens that are making them sick. 

The solution that I'm working on is actually making this flagellin receptor recognize different flagellins that right now, the plant is unable to recognize. I'm just going to walk you through the steps of what I've actually done in my research to try to and help these plants recognize some of these peptides from different bacteria. The first step is to actually create new versions of this receptor. I mentioned before that the FLS2 receptor sort of looks like a Slinky in mid-tumble down the stairs. It's sort of a spiral with a nice little curve in the middle that you can imagine flagellin coming in and binding to quite nicely. We're quite certain that when the receptor recognizes flagellin, it actually is recognizing it right here in this curved region. So what we do, is we make mutations in this region, in the hope that it will allow the receptor to recognize different flagellin from different species. Once we've created those different versions, and when I say create different versions, I mean that we actually clone a gene, and then are going to put that gene into the plant. So Arabidopsis, as I mentioned earlier, are very amenable to genetic transformation. The way that we do genetic transformation is through what I think of as a reformed plant pathogen that used to do evil, and now we actually use them for good. 

It's called agrobacterium. What agrobacterium does in the wild, is it will create what's called Crown Gall Disease, which is basically it's like cancer for plants. It causes uncontrolled cell growth. So if you've ever seen like a gall or a knot that's growing on a plant, sometimes that's caused by species that are related to agrobacterium.  When they cause disease, basically what agrobacterium is doing, is it's actually taking a piece of DNA and putting it into the plant genome, to actually cause the plant to develop some of these gall or tuber-like structures. By the way, if anyone can read Spanish, great, you can read the captions! Otherwise, I'll walk you through it. But in the case of when we use this for plant transformation, what we've actually done is instead of having the agrobacterium put in its own nefarious genes that cause uncontrolled cell growth, we have it put in our gene of interest. In this case, the gene that encodes for the FLS2 receptor. So, we actually just take plants and dip them in a solution of agrobacterium, which I can tell you smells. I won't tell you how it smells. 

But the point is that once we do that, we can then go and harvest the seed from the plants that have been dipped in this agrobacterium stream. This is what Arabidopsis actually looks like when it's flowering. These are the seed pods growing off of that stalk in the middle. We can go and harvest the seeds out of that seed pod, plant those up, and then look for ones that actually got our new version of FLS2 in them. The way that we do this is by a selection mechanism. What we do is, in addition to having the agrobacterium insert our interesting gene, we also put in what's called a marker gene. So what a marker gene does is it basically is a way of us marking the plants that got our gene of interest in them. In this case, this is an herbicide resistance gene that we put in. What we do, is we grow the plants on herbicide, and only the plants that have an herbicide resistance gene are able to actually grow on these plates. So that's illustrated here. 

It's a bit difficult to see, but these bright green plants represent what the herbicide resistant plants actually look like when we grow them on these petri dishes with herbicide. So, bright green plants mean they're herbicide resistant, mean they have our new FLS2 gene in them, mean that we're interested in studying them. I take these plants and actually move them into a plate that we're going to actually study the interaction of these FLS2 receptors with the flagellin. So, just to quickly remind you that the assay we're doing is a seedling growth inhibition assay, we're going to be exposing the plants to flagellin and then looking to see if they're smaller when we expose them. If plants are smaller when we expose them to flagellin, that means they're actually able to sense the presence of that molecule. So what we're hoping to see is something like this, where after a couple weeks of growth, plants that have not been treated with flagellin grow much larger than ones that have been treated with flagellin. What's wonderful about plants in this assay is that it's really easy to do. We just weigh them. It's non-invasive. So after we're done, after we've weighed them, we can save them to do other types of assays, to double check to make sure that yes, they really are able to respond to these flagellin molecules. 

These eight bars summarize two years of work. What you can see here is we have on the left, a wild type receptor that you would find in a plant just outside our door. What you can see is if we grow them, either with or without, so 1/2MS is just the media they grow in. If we grow them with or without this flagellin peptide, they are basically the same size. However, I've been able to find three different versions of FLS2 that actually, when you grow them with this fire blight flagellin, the plants are responding. You can see that they are actually smaller in the presence of this flagellin. Just to conclude, what we've come up with here is the first step toward helping plants resist infection. We have a plant that's actually able to recognize pieces of a bacteria that it wasn't able to recognize before, which is really, really exciting, because we hope that this can actually lead to plants that are more resistant to this pathogen. The graph I basically just showed you is that this plant is now able to recognize a flagellin from a pathogen that previously it couldn't sense and could cause disease. Now the question is, can we go back to the trees? Can we actually put this into a crop species and actually make the plant more resistant? Because, as you might expect, although there are many similarities between Arabidopsis and trees, Arabidopsis plants are not apple trees, so it's very important to check to make sure that these receptors actually work in the species we're interested in. 

We do know from previous experience that this can actually work. What I'm showing you here are tomato plants, sort of an aerial view looking down on top of them. What you can see is that these ones on the left look rather pathetic compared to the ones on the right. Both of these plots of plants were infected with a bacteria, the same bacteria. The difference is that the plants on the right contain a single receptor from Arabidopsis, a single immune receptor from Arabidopsis, I should say. So just having the presence of one additional receptor in these plants allows them to be far more resistant to bacterial infection, and allows them to be much healthier. Just to conclude, I want to quickly thank the members of my lab. We work over in Russell Lab, which is just a couple blocks down the street. Our funding sources, Department of Energy grant, and a National Science Foundation Fellowship to myself. The members of my thesis committee, collaborators we work with, and the plant cell and the bacteria cell would also like to thank you for your attention. 

I thank you for your attention, as well. I'd be happy to answer any questions that you have. [APPLAUSE]