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.