Session 8: Plant Immunity and the Evolutionary Arms Race between Host and Pathogen
Transcript of Part 3: Arabidopsis thaliana-Pseudomonas syringae interaction: The effect of climate in plant disease
00:00:07;22 Hello. 00:00:08;22 I'm Sheng Yang He. 00:00:09;23 I am a professor at Michigan State University and an Investigator at the Howard Hughes Medical Institute. 00:00:15;24 This is Part 2 of my iBiology talk. 00:00:19;08 In this part of my talk, I want to tell you some of our work involving Arabidopsis and 00:00:27;06 the Pseudomonas syringae interactions. 00:00:29;06 Particularly, I want to highlight one aspect of our research, illustrating how environmental conditions 00:00:34;24 could profoundly influence disease development in plants. 00:00:39;21 So, as you know, when you look at a plant growing in nature, outside, they not only are, 00:00:47;07 you know, exposed to potential pathogens, but they are also experiencing a lot of different conditions: 00:00:52;24 temperature fluctuation, you know, from morning to evenings; light, as you can see here; 00:00:59;20 and temperature; humidity; and microbiome, even. 00:01:03;23 We know that all of these factors actually influence pathogen and plant interactions. 00:01:09;09 The molecular bases of this are not well understood. 00:01:12;24 And so some famous scientist said, you know, without understanding environmental conditions, 00:01:16;26 we will never understand the immunity in plants... you know, in the plant system. 00:01:23;04 So, I'll just give you a couple of examples of how important the climate conditions could be 00:01:28;00 for plant disease outbreak in the field. 00:01:32;11 This is the bacterial fire blight disease in apple. 00:01:36;11 This is in Switzerland. 00:01:37;11 This is a 12-year span of disease incidents from 1995 to 2007. 00:01:45;18 So, apples are always growing in, you know, Switzerland, and pathogens are always in these orchards, 00:01:50;20 but you don't see the disease every year. 00:01:53;15 And the reason is for disease to occur you need a lot of humidity and the right temperature, right? 00:02:01;10 So, in 2007, in that year you have heavy rain and high humidity in the spring, 00:02:05;23 when the apple was flowering, and these bacteria tend to infect the flowering parts. 00:02:11;19 And so everything kind of came at the same time, and then have very severe disease. 00:02:16;17 So, that's one example. 00:02:19;08 Another example is called fusarium head blight of wheat. 00:02:23;07 This is actually a very huge global disease right now. 00:02:28;11 It's also favored by high humidity and warm temperatures in the spring. 00:02:31;22 So, you can see that... you know, normally you see a nice green top of the wheat. 00:02:38;03 In this image, you can see, basically, bleached grains. 00:02:43;15 And there were four very severe epidemics in China in the last five years, 00:02:47;28 so almost every year has very severe disease. 00:02:49;25 This disease, also, is very serious, because the fungus actually produces a toxin 00:02:56;11 which makes us sick. 00:02:57;11 And so... not only reducing yield, but also causing sickness in the human population. 00:03:05;15 So, I want to tell you that plant diseases are really, you know, problems in modern agriculture. 00:03:11;19 They're really major threats to food security, globally, right now. 00:03:16;19 Some of these diseases are very old. 00:03:18;28 On the left is a disease called rice blast, a disease I actually grew up, when in China 00:03:25;04 I lived in a village with 200 people or so. 00:03:29;04 So, you know, I saw this rice blast when I was a really small little child. 00:03:35;01 When I go back right now, 40 years later, and talk to my parents, and this is 00:03:41;04 still the number one disease locally, but also globally, in rice production. 00:03:45;06 So, many old diseases continue to really pose major problems. 00:03:49;10 Now, you also have new diseases coming up. 00:03:52;20 One example I'm giving to you here is a kiwi bacterial canker, which is caused by 00:03:57;14 a bacterial pathogen called Pseudomonas syringae, and I'm going to tell you a little bit about that today. 00:04:02;12 So, this is despite all the chemical input -- you know, pesticides, you have to spray them, 00:04:08;09 farms have to use them, because otherwise you won't have, you know, really high yield -- 00:04:11;20 but also all the breeding efforts, that many scientists try to breed resistant cultivars. 00:04:18;21 You know, from wheat to rice, based on these so-called disease resistant genes. 00:04:25;03 But this is not enough. 00:04:26;03 So... because we have disease every year still. 00:04:28;18 One of the problems, as we've realized, is that we really don't understand 00:04:32;05 the basic process of disease, okay? 00:04:34;16 So, this is an area that we really want to push ahead. 00:04:39;02 So, in the last 15 years or so, you know, many laboratories including us are 00:04:45;11 really concentrating on trying to work out why disease occurs. 00:04:49;22 And so this is an overview of different kind of pathogens that can cause disease in plants. 00:04:54;19 So, we have fungus; we have bacteria; we have nematode, worms, you know; and viruses. 00:05:03;02 Many of these pathogens also cause problems in our human bodies also. 00:05:06;25 And so one... so, they look very different, but one of the common things they do is to 00:05:10;18 deliver these virulence factors -- collectively, we call them effectors -- into the plant cell. 00:05:16;27 And... and so, they use different ways of delivering these virulence proteins. 00:05:21;20 In the case of bacteria, they use a secretion system called the type III secretion system. 00:05:26;02 You can see on the right a syringe-like structure, here. 00:05:31;08 If you knock out this delivery system, bacteria become non-pathogenic, okay? 00:05:35;22 So, that illustrates how important these virulence factors are to causing diseases. 00:05:41;06 So, because of that, studying how effectors work really can provide great progress into 00:05:48;25 the molecular basis of disease susceptibility. 00:05:52;27 And interestingly, these molecules, microbial molecules, also can be very powerful probes 00:05:58;19 into the fundamental biology of the host -- and that can be plants or it could be humans -- 00:06:03;13 because they usually find very intriguing RNAs or proteins or DNAs to manipulate the host physiology. 00:06:09;27 Okay, so in a sense, this is a really great, you know, probe into the biology of the host itself. 00:06:16;08 Obviously, discovering the target of these virulence factors could offer new leads into 00:06:21;24 innovative disease control we really desperately need right now. 00:06:25;25 So, how do we understand disease susceptibility? 00:06:28;07 Which approaches? 00:06:29;23 We and others are really following this very simple diagram, here. 00:06:32;24 We want to understand the host target of all these bacterial virulence proteins. 00:06:38;17 So, in the case of the bacteria we study, it has about, you know, 30 or so effectors. 00:06:43;04 What we want to do -- we means us and the many other laboratories -- is really to identify 00:06:48;12 these host proteins or RNAs or DNAs that are being targeted by these virulence factors, 00:06:54;12 and we want to associate these host targets to these particular pathways. 00:06:58;05 You know, I listed the five of them -- A, B, C, D, E -- but it could be 30, right? 00:07:02;28 So, we don't know how many pathways are being targeted by bacterial virulence factors. 00:07:08;00 What we hope to do is to... once we identify these pathways, we could genetically 00:07:14;00 perturb these pathways in the host, in this case, in the plant. 00:07:17;25 And if we're successful, then if we understood everything about the disease process, 00:07:23;13 we can create a poly mutant of the host in which these pathways are basically either 00:07:29;16 activated or inactivated to simulate the collective activity of these virulence factors. 00:07:36;03 And then if we really understood the process then, then the poly mutant of the host 00:07:40;05 would be susceptible to a bacteria that is not able to produce effectors. 00:07:44;22 In other words, if we manipulate the host already, genetically, to simulate the 00:07:50;17 action of the virulence factors, you don't need these virulence factors to start with, right? 00:07:53;28 Until then, we will never know we understood the disease, okay? 00:07:56;27 So, that's the goal. 00:07:58;06 It's very challenging, but by the end of these twenty minutes, I want to show you that 00:08:02;05 we have made progress towards that goal. 00:08:04;28 So, we use this very simple model system involving Arabidopsis, which is a model plant, 00:08:12;04 and a bacterial pathogen called Pseudomonas syringae. 00:08:14;21 It's a very common pathogen. 00:08:16;13 It infects virtually all crop plants in the field, okay? 00:08:20;25 Each individual string of this species, Pseudomonas syringae, infects a very narrow range of hosts. 00:08:27;02 So for instance, strain DC3000 in the field only infects tomato. 00:08:32;24 In the laboratory, you can also make it infect Arabidopsis, okay? 00:08:37;05 So... so because Arabidopsis is a very, you know, powerful model for plant research... 00:08:42;05 so we have been working on Arabidopsis-Pseudomonas model system for many years now. 00:08:48;00 Pseudomonas can actually live on the surface of the bacteria... of the plants as an epiphyte. 00:08:54;15 But in order to cause disease, it has to go into the interior of the leaf, in this case, okay? 00:09:00;20 They go into the leaves through so-called stomata. 00:09:03;16 So, these are microscopic pores on the leaf epidermis that allow plants to take up 00:09:09;15 the CO2 to make food for us, okay? 00:09:12;02 So, photosynthesis. 00:09:13;02 It's very important, okay? 00:09:15;22 And once bacteria go into the... inside the leaf, it lives in between the cells, okay? 00:09:21;09 So, these are called mesophyll cells. 00:09:23;12 So, these are extracellular pathogens, okay? 00:09:25;21 So, this space is called the apoplast. 00:09:27;06 Now, I want to tell you the apoplast is normally filled with air. 00:09:31;13 It's not filled with liquid. 00:09:32;13 This is very important, because CO2 has to go into the... goes through to the stomata 00:09:37;18 into the apoplast, but it has to diffuse into the mesophyll cell and the chloroplast. 00:09:42;04 So, it's a long distance for the CO2 to go in there. 00:09:45;03 You don't want water in there, because there will be very high resistance to CO2. 00:09:51;00 So, the plant has a way of keeping that space mostly filled with air. 00:09:55;04 I'll come back to this. 00:09:56;04 It's actually very relevant to pathogenesis. 00:09:58;22 So, what we do in the laboratory to kind of have a disease assay is really to grow plants 00:10:04;08 in a pot. 00:10:05;25 You probably do this at home. 00:10:07;14 Not this style, but in another way. 00:10:10;01 And then, when they are four or five weeks old, we would dip the plants entirely 00:10:15;19 into the bacterial suspension and wait for, basically, three days, okay? 00:10:19;11 You will see disease symptoms, as shown here. 00:10:21;15 So, I'm gonna play a movie which shows you the time-lapse video of the infection process. 00:10:28;05 On the left are the mock... 00:10:29;19 I mean, are the bacterially infected plants. 00:10:33;04 On the right is a mock infection, this is water, okay? 00:10:35;24 So, what you can see, now... eventually, you can see the yellowing on the plants that are infected. 00:10:42;00 And on the right are the ones that are moving, you know, they are alive, okay? 00:10:45;17 You can see there are some plants that are kind of dancing, of this thing you can see. 00:10:50;12 But the infected plants are basically paralyzed, okay? 00:10:53;02 So, we actually don't know why plants are motionless very early on in the disease. 00:10:58;00 This is one of the things we are trying to understand in the... in the next few years. 00:11:02;18 So, we have worked on several aspects of this disease process. 00:11:08;14 For instance, we have, a few years ago, figured out that entry process, right?, how bacteria 00:11:15;07 enter the plant tissues through the stomata. 00:11:18;25 For a long time, scientists think they are passive, because the stomata pores are 00:11:24;11 quite big and bacteria are kind of small. 00:11:25;26 They can... the pore has to be open for photosynthesis during the day, so we always thought bacteria 00:11:31;11 can just take advantage of that and go into the tissue, like, passively, right? 00:11:34;17 That doesn't turn out to be the case. 00:11:37;00 It turns out these guard cells -- there are two guard cells to form one stomata pore -- 00:11:42;18 they actually can sense bacteria. 00:11:45;03 And so once they sense the bacteria, they close it as the first line of defense, 00:11:50;02 to prevent any microbes entering the tissue. 00:11:52;27 So, plants are very smart, okay? 00:11:54;12 So, that's about... a very intriguing mechanism defending against pathogen invasion. 00:12:00;23 We discovered one of the... so, that's bad for the bacterial pathogen, right? 00:12:04;00 It cannot even start the infection. 00:12:05;12 So, in the case of Pseudomonas syringae, it figured out a way to prevent that from happening 00:12:10;02 by producing a toxin called coronatine, which prevents stomata from closing. 00:12:15;07 And so the bacteria can massively infect to start an infection. 00:12:20;14 Once the bacteria get into the mesophyll space... as I mentioned before, it's an extracellular pathogen, 00:12:25;13 but it makes a type III secretion system injecting more virulence effectors 00:12:30;01 into the plant cell as a major weapon of pathogenesis. 00:12:32;28 So, we're working on this area as well. 00:12:35;28 So, we knew a little bit of these basic steps of this infection involving stomata entry, 00:12:41;13 involving a toxin that prevents the stomata from closing, and involving these effectors 00:12:46;17 that we think, now, are suppressing immune responses in plants, okay? 00:12:51;07 Work in the past few years, from us and many other groups, has deepened our understanding 00:12:56;26 of these basic steps, but also... in our case, we realized that we're missing two dimensions 00:13:02;21 in the last, you know, many years, actually. 00:13:05;00 One dimension involves the profound effect of environmental conditions on the host-pathogen interactions. 00:13:10;19 So, that's under the left circle, here. 00:13:14;13 We also started to realize the endogenous microbiome -- the plant also has a microbiome -- 00:13:19;00 has tremendous effect on host-pathogen interactions. 00:13:21;21 So, these are new directions. 00:13:22;27 I'm gonna highlight one particular area, which is involving how environmental conditions 00:13:28;17 could influence the disease interactions, okay? 00:13:31;24 So, we are focusing on two areas. 00:13:34;27 One is the temperature, how elevated temperatures could influence disease. 00:13:39;09 This is actually very relevant right now with climate change. 00:13:42;16 The globe is warming. 00:13:44;11 But also, more importantly, the heat waves we're experiencing in different countries 00:13:49;06 are very severe right now... and how these short periods of heat waves could influence infection. 00:13:55;26 Okay, so this is one of my students, Bethany Huot, who recently published a paper 00:14:00;20 just showing very simply... you can see under... we grow plants the same way, okay?, 00:14:05;15 but during infection we put the plants in 23 degrees, which is the normal temperature, or you shift 00:14:11;04 5 degrees up, to 28 degrees, you can see dramatic differences already. 00:14:15;08 At the warm temperature, you see much more severe disease, okay? 00:14:19;20 She discovered this is based on two mechanisms. 00:14:22;21 One is the warm temperature actually enhances greatly the virulence expression. 00:14:26;28 So, the effector secretion into the plants is greatly enhanced. 00:14:32;05 But also, she discovered that the immune signaling in the host is completely shut down. 00:14:37;21 So, this is actually very important in the field, you know. 00:14:40;21 The immune pathway that she was working on is called salicylic acid signaling, 00:14:45;28 which is mimicking, like, the aspirin we take sometimes. 00:14:48;00 It's a similar chemical. 00:14:49;14 It boosts the immune response. 00:14:51;17 This response is shut down by warm temperature. 00:14:54;15 This could have a profound influence in the field, crop resistance, because most of the 00:14:58;28 crop resistance is based on their signaling cascades. 00:15:01;12 So, we don't know the details of this pathway. 00:15:03;24 This is something we're gonna work out in the next few years. 00:15:05;24 What I'm going to talk to you about in more detail is humidity's effect on plant disease, 00:15:11;02 okay? 00:15:12;02 This became, actually, obvious in our disease reconstitution experiment I mentioned in the 00:15:15;12 beginning of my talk. 00:15:16;21 We tried to figure out how many pathways are being manipulated by the bacterial pathogen. 00:15:21;18 And ultimately, we want to create a poly mutant of the plant to see whether we can 00:15:26;21 rescue the pathogenesis of a bacteria that does not, you know, deliver any of these effectors, 00:15:31;12 okay? 00:15:32;12 So, that's a very daunting task, but we... as scientists, we want to, you know, 00:15:37;08 face the challenge and try to work it out. 00:15:39;22 So, there are 30 of so effectors, I told you, in this particular bacterium, so we and others 00:15:45;03 are systematically going through to identify the host target of each of these effectors, 00:15:49;16 okay? 00:15:50;16 A model that we and others have developed in the last, you know, 15 years or so about 00:15:55;25 the function of these effectors is this, in a simple way. 00:15:58;27 So, you're seeing a bacteria sitting on the plant cell wall. 00:16:02;10 So, a plant cell, unlike an animal cell, has a cell wall surrounding it. 00:16:05;18 But in the plasma membrane, which you can see, there are receptors. 00:16:09;17 They're called immune receptors, that perceive these patterns from microbes, in this case, 00:16:14;16 flagella, these wavy things, very common for bacteria. 00:16:18;07 And once they sense these molecules, it then triggers a signal transduction pathway 00:16:22;20 -- this is a very simple diagram -- eventually leading to a form of immunity called pattern-triggered immunity. 00:16:29;06 So, this is bad for bacteria, so what bacteria are doing is to send these effectors 00:16:34;06 into the plant cell to attack different steps of this signaling cascade, to shut down 00:16:38;24 this form of immunity. 00:16:39;24 It's a major mechanism of disease. 00:16:43;05 And so, I'll just give you one example from a collaborative work from Cyril Zipfel's group 00:16:48;11 and my laboratory, also, involving a particular effector called HopAO1. 00:16:53;01 HopAO1 biochemically is a phosphatase, which removes phosphate from proteins. 00:16:59;22 And it turns out these immune receptors are phosphorylated, normally, during activation 00:17:04;14 at a tyrosine residue of the protein. 00:17:07;11 And this effector actually removes the phosphate from tyrosine to shut down this immune activation. 00:17:12;02 So, this is a very cute way of... you know, bacteria figured out how to kind of sabotage 00:17:17;14 the immune signaling. 00:17:18;18 And there are many studies to support this, very strong evidence that this is really true. 00:17:23;10 So, one of the major functions of these virulence factors is to shut down the plant immune response, right? 00:17:29;10 If there's no immunity response in the host then you can, you know, infect the plants. 00:17:32;16 And this is very similar to human pathogenesis. 00:17:35;03 And many of the bacterial pathogens are human pathogens that actually do the same thing. 00:17:39;02 They're shutting down the immune system in our body, then infect. 00:17:42;22 Okay, so our question is this. 00:17:44;20 Are all these 30 or so effectors involved in immune suppression? 00:17:48;06 If they're all attacking, you know, immune suppression, then we can reconstitute the disease 00:17:53;23 by using the immune compromised plants, right? 00:17:56;27 So, I'm coming back to point... that point later. 00:17:59;13 I've introduced you to two bacterial strains, now. 00:18:02;10 I'm talking about the wild type strain, DC3000. 00:18:05;17 It secretes these 30 or so effectors into the plant cell. 00:18:08;13 There's a mutant called delta-28E, which has 28 of these 30 effectors deleted. 00:18:14;27 It has involved a lot of work done by Alan Collmer's lab at Cornell University, 00:18:21;02 but they did it, so it's a very useful mutant, and we take advantage of this mutant. 00:18:25;20 Because this mutant has essentially no effectors that are delivered into the plant cell, 00:18:30;04 it's not pathogenic. 00:18:31;04 So, if you put into a wild type plant... you can see that on the left is infection by DC3000. 00:18:36;12 It causes disease-like symptoms. 00:18:38;09 But on the right is green; it's a healthy plant. 00:18:40;28 So, this mutant cannot cause disease in the wild type plants. 00:18:45;22 If, as I said, all effectors are attacking the immune signaling, then if we start with 00:18:51;22 immune defective plants, if there's no immunity in the plants, then this mutant, delta-28E, 00:18:58;27 should be able to infect the plants, right? 00:19:01;22 Okay, so that's the experiment we did. 00:19:03;23 You can see that, unfortunately, the delta-28E mutant was unable to cause disease. 00:19:09;14 You know, the plants are still kind of green after infection, okay? 00:19:13;04 The mutants we used, fec and bbc, these are defective immune responses in the plants. 00:19:18;16 So, the answer is no. 00:19:19;28 Okay? 00:19:20;28 So, you can also look at the bacterial population. 00:19:22;21 So, when the plants are infected by Pseudomonas syringae, it multiplied really high. 00:19:27;17 So, this is... the bar is in the logarithm... log-type scale, so each step is a tenfold increase. 00:19:36;04 You can see that DC3000 aggressively multiplied inside the leaf. 00:19:40;20 Versus the delta-28E in wild type and in mutant leaves, they are unable to achieve 00:19:47;10 a very high population. 00:19:48;10 So, there's no disease, so the answer is no. 00:19:50;05 So, the question is, what are we missing?, right? 00:19:52;26 So, some effectors must be attacking something other than immunity as a part of their mechanism. 00:19:59;02 So, I'm gonna pull you away from my... our own results to tell you something about a website. 00:20:05;22 So, if you're growing plants in your garden, this is actually for master gardeners, 00:20:10;09 so anything written on this website must be true because you have to follow that. 00:20:14;08 Okay, so you can see that I just took a few sentences out. 00:20:18;05 It says, bacterial diseases are most intense in warm and humid conditions like Florida, 00:20:23;26 okay? 00:20:24;26 So, Florida actually has a lot of diseases compared to California. 00:20:26;26 California is dry. 00:20:30;02 You can recapitulate... this is actually famous idea called the "disease triangle" dogma. 00:20:34;17 For a disease to occur, you not only need a planet which is susceptible genetically 00:20:38;06 and a pathogen which would is virulent genetically, but you also need a conducive environment. 00:20:42;26 One of the main factors is high humidity, okay, rains and things like that. 00:20:47;27 This was formulated by a very famous plant pathologist, RB Stevens, 50 years ago. 00:20:52;14 We actually don't know the molecular basis by which humidity is required for disease 00:20:56;03 very much. 00:20:58;08 You can recapitulate the humidity requirement in the laboratory. 00:21:01;07 Basically, you can grow plants, you know, for four weeks. 00:21:04;12 But during the infection period of three days, we either place the plants under high humidity, 00:21:09;13 like 95% percent, which simulates the disease outbreak condition in the field, or you, 00:21:15;02 you know, set up the plants at [30%], which is a low humidity. 00:21:19;20 You can see that at high... and only at high humidity you have disease. 00:21:22;16 At low humidity, plants look healthy. 00:21:25;11 And you can look at the disease bacterial population, also. 00:21:29;08 High humidity has a very high population, and under lower humidity you have very low. 00:21:34;20 Okay, so it's a dramatic difference, okay? 00:21:36;24 Now, if you go back to this website, you can also see a term called water soaking. 00:21:42;02 This is describing the symptom of the disease of many bacterial diseases. 00:21:45;24 Normally, if you look at leaves in your backyard you will see kind of, you know, green, okay? 00:21:51;15 There's no spots, right? 00:21:53;15 In this picture, you can see there's a lot of dark spots. 00:21:55;18 These dark spots are caused by liquid in the leaf. 00:22:00;17 And plants don't like that. 00:22:01;17 I just how you been beginning, for photosynthesis to occur, for CO2 to diffuse into the mesophyll cell, 00:22:06;10 you want to keep the apoplast air-filled. 00:22:09;20 And in these dark spots, there's liquid in there. 00:22:12;12 It's really bad for plants. 00:22:13;21 But bacteria seems to be able to do this for a purpose. 00:22:17;00 We are actually... so, phenomenon has been observed for many decades. 00:22:20;22 We don't know whether it's needed for pathogenesis, okay? 00:22:24;07 So, we were intrigued by this. 00:22:26;11 This only occurred under high humidity, also. 00:22:28;13 So, you can simulate this process in the laboratory. 00:22:31;24 This is our Arabidopsis, again, infected by Pseudomonas syringae. 00:22:36;10 You can see dark spots, here, on the right leaf, which is infected. 00:22:39;19 On the left, that was not infected. 00:22:41;25 This also occurred in tomato, because this bacteria also infected tomato. 00:22:45;01 So, under high humidity, you have this so-called water soaking symptom. 00:22:49;25 Now we can label bacteria to see where the bacteria are in the infected tissue by 00:22:55;18 loading it with a luc... you know, lucs emit light... allow bacteria to emit light. 00:23:00;28 So, you can catch the light emitted from bacteria in the infected tissue and then overlay this 00:23:07;25 with the water soaking symptom that you capture with regular light. 00:23:10;26 And if you see, in the bottom of the left leaf, we can see extensive overlap 00:23:16;18 between the luc -- the light indicating bacteria -- and the water soaking spots, suggesting that 00:23:24;17 the water soaked area is where bacteria multiply really highly, okay? 00:23:27;23 So, that is really spatially kind of indicating water soaking is quite important. 00:23:32;23 So, what causes the water soaking? 00:23:35;02 Okay, I told you this bacteria produces 30 or so effectors. 00:23:38;14 We actually screened each individual effector to see which one can cause water soaking. 00:23:43;14 In this experiment, we show that two of them can cause water soaking. 00:23:47;16 And the names are not very important, but I can show you that one is localized to 00:23:51;20 the plant plasma membrane, here. 00:23:54;01 One is localized to, actually, the endomembrane system in the plant cell, called the endosome, 00:23:58;26 which is involved in recycling all the proteins to and off the plasma membrane of the plant cell. 00:24:05;03 So, they're two... these two effectors are doing something to the plasma membrane 00:24:08;06 of the plant cell to cause water soaking. 00:24:11;24 We actually know a little bit more about one of these effectors. 00:24:14;01 They actually attack a protein in the plants that regulates the vesicle traffic. 00:24:19;02 So, it's a really intriguing phenomenon, also, because a lot of human pathogens also do that, 00:24:25;04 attack proteins that are involved in vesicle trafficking in our human cells as a way to 00:24:29;23 shutting down the immune system. 00:24:32;01 Okay. 00:24:33;01 So, now we have... in addition to the immune suppression process, we discovered a new process 00:24:38;14 we called aqueous apoplast, which is the inside of the leaf accumulating, basically, 00:24:43;17 water and other things. 00:24:45;11 Okay? 00:24:46;11 So, in order to cause water soaking, you need the so-called water soaking effectors 00:24:50;26 from bacteria. 00:24:51;26 But that's not sufficient. 00:24:52;26 You also need high humidity in the air. 00:24:55;23 The reason is that in the low humidity, even if the bacteria are producing water soaking symptoms, 00:25:01;05 it will be evaporated out through stomata, because stomata are open during the 00:25:04;24 day for... to take up CO2. 00:25:07;22 And because of that, if you have low humidity, the water just comes right out. 00:25:11;18 And because there's no water, then the bacteria will not benefit. 00:25:14;16 So, here's an example where we need variance factors in the bacteria and we need 00:25:18;18 the external environment to be humid, okay? 00:25:20;05 So, this is kind of interesting. 00:25:22;28 So now, the question. 00:25:23;28 The next question we want to ask is... okay, we have two processes now. 00:25:27;11 We know that immune suppression is not sufficient for pathogenesis. 00:25:31;00 Now we have two... are they sufficient, now, for pathogenesis? 00:25:35;00 So, this is a disease reconstitution experiment we always wanted to do. 00:25:39;15 So, we can simulate the suppression of the immune response in the plant by using 00:25:46;14 this mutant of Arabidopsis that is unable to mount an immune response. 00:25:50;00 We can also mimic the water accumulation in the apoplast by using this new mutant 00:25:55;06 that we have, called min7, okay? 00:25:57;08 The idea is to combine these two process by genetically manipulating the two pathways. 00:26:03;07 Using CRISPR/Cas9 technology, we created quadruple mutants, basically affecting both immunity 00:26:09;26 and water homeostasis. 00:26:11;27 So, the question is that, in these quadruple mutants, would bacteria that normally 00:26:17;25 cannot deliver any effectors... is going to multiply or not, okay? 00:26:22;02 So, this is the experiment we did. 00:26:24;09 So, the bacterial mutant we use is the bacteria that are unable to secrete any of these effectors, 00:26:28;25 that's defective in type III secretion, okay? 00:26:31;06 In the wild type plants, they don't cause disease. 00:26:34;06 It's green plants. 00:26:35;27 In these immune-defective mutants, it still does not cause disease, as I showed you before, 00:26:41;06 okay? 00:26:42;06 So, it's not sufficient. 00:26:43;13 In a min7 plant, also, it does not cause disease. 00:26:46;17 In the quadruple mutants, now, you can see disease-like symptoms. 00:26:50;11 And this is actually when we're seeing a non-pathogenic bacteria cause any disease on a plant system. 00:26:56;19 So this is pretty exciting to us. 00:26:58;24 If you look at the bacterial population in these leaves, you can see that the red bars 00:27:03;10 are indicating the quadruple mutants. 00:27:05;15 Only in these two quadruple mutants, you can start to see the multiplication of an otherwise 00:27:10;11 non-pathogenic bacteria, okay? 00:27:12;17 So, it's not to the extent of the totally wild type infection, so we have some distance to go, 00:27:16;08 but this is a quite significant step. 00:27:18;25 So, summarizing this part of my talk, we have identified a new pathogenic process involving 00:27:25;27 what we called aqueous living space. 00:27:29;04 We know bacteria loves water because, you know, human pathogens and plant pathogens 00:27:34;05 all love water, right? 00:27:35;13 So... but this is a case where bacteria actually create water conditions in an otherwise air-filled space. 00:27:43;22 And if you think about whether this is relevant to, you know, other diseases, 00:27:47;09 including human diseases like a lung infection and the respiratory system, which is normally filled with the air... 00:27:53;12 so, we will see whether this principle will go beyond plant diseases, okay? 00:27:57;17 We were able to reconstitute the basic features of a bacterial infection within 00:28:03;02 exclusively host mutants, okay? 00:28:05;03 So, that's also the first time we've done this. 00:28:08;02 Of course, we're getting some insight into why humidity could have profound influence 00:28:12;08 on the disease interactions. 00:28:15;13 In this case, because it's required for the virulence factors to function as virulence factors. 00:28:21;04 So now, I'd like to acknowledge the people that actually did the work. 00:28:24;07 Of course, my lab members at Michigan State. 00:28:28;01 And I also want to acknowledge a number of collaborators: Jeff Chang, Cyril Zipfel. 00:28:34;25 Also other investigators that I collaborated with for the other part of my talk. 00:28:40;24 Funding are from HHMI, Gordon and Betty Moore Foundation, NIH, DOE, USDA, 00:28:46;24 and the National Science Foundation. 00:28:49;02 Thank you.