Skip to main content
Loading Explore guides…
Plant-Pathogen Interactions · Acedra
Home
Explore
Biology
Plant-Pathogen Interactions
Sign in to save
Chat
If YouTube asks you to sign in, open the video on YouTube.
Watch on YouTube
Reload player
Transcript
100 segments
Copy
Hide video
0:03
Hi, I’m Jonathan Jones. I’ve got a talk coming up called, “Pathological intimacy: how microbes attack plants and how plants fight back”. So I’ll be telling you about kinds of plant diseases; I’ll be telling you about kinds of ways in which pathogens attack and get into plants; and I’ll tell you about various levels of resistance mechanisms that enable plants to detect pathogens and turn on the appropriate defences.
0:39
Thank you. It’s a pleasure to be here. It’s wonderful to have a chance to meet the next generation of leading plant scientists, which is, I’m sure, what you all are here. But it’s a very daunting task, because I don’t normally lecture to undergraduates so I’m guessing about what level to pitch it at. But the most important thing I’m going to be trying to do is to get across certain absolutely key concepts in trying to understand how plants resist disease
1:11
and how microbes circumvent that resistance. So here… this may look a bit of a tangential thing to refer to, but you’ve all heard of the sensitive plant, and here’s a sensitive plant. And it’s wonderful to behold, and of course it’s got this name: the sensitive plant.
1:32
I always find this slightly irritating, because in fact all plants are sensitive plants. Plants perceive what’s going on in their environment and adjust their growth accordingly, and they also turn on a bunch of responses to help them resist disease where they’re under attack, from diseases or also from pests. So my way of getting across some of the key concepts in the field… here’s the points I’m going to attempt to make. I’m going to tell you
2:05
about plant disease in a fairly general sense and the types of plant pathogens, and then I’ll get into the various questions you may have about plants and their interactions. So I’m going to start off with how pathogens get in and start causing a problem, and then I’m going to talk about resistance mechanisms, and also say a little bit about this plant pathogen, simply because it’s so important. And there’s a lot of other science of plant-microbe interactions which I’m not going
2:31
to get into, like systemic acquired resistance or jasmonate signalling for insect resistance, just because you can’t do everything. But a lot of the content in what I’m going to be telling you, you can find in a chapter that I contributed with Kim Home and Kozak to this book, and also the key concepts can be found in this book which I just happened to see down there on the list of the set of books that you’ve got. So chapter eight in here will also cover
2:58
a lot of the content that I’m going to go through if you want to read more about it, or if there’s something you felt you missed that was interesting and you’d like to get clear. First of all with plant-microbe interactions in general… so not all interactions between plants and microbes result in disease. Of course, this is a disease example here. Over here, you’ve got some more classic symbioses, so rhizobium bacteria colonise roots of legume plants and
3:32
make these nodules within which nitrogen fixation can occur. This is particularly advantageous to plants on nitrogen-depleted soils. And then there are these fungi called arbuscular mychorrhizae, which have a very intimate relationship with plants and facilitate their uptake of phosphate. And there are some other more curious examples. So epichloë is a fungus that grows in rye grass, and it seems that by making a lot of secondary products that are poisons for animals, it actually
4:07
deters browsing of animals on the plant. So this is essentially a symbiotic interaction, but it’s a big problem for sheep farmers in New Zealand because lolium gets colonised by these epichloës that make compounds that are actually rather toxic to sheep. But nevertheless, not all plant-microbe interactions result in disease, as in this example here of rice blast on rice. But one of the more important diseases of plants
4:35
is this late blight caused by Phytophthora infestans that affects potato and tomato. And the remarkable thing about this disease is that, from a small colonisation within a field, within a couple of weeks a field can be just completely wiped out and turned to toast.
4:58
So, an important thing to know about this particular type of organism is it’s not a fungus. You’ll often see it in newspapers: “the fungal disease Phytophthora infestans”, late blight. Actually, if you look on this tree of life here… so here’s phytophthora. It’s more closely related to brown algae and to things like malaria or even to plants than it is to fungi. In fact, humans are more closely related to fungi than phytophthora is. And we feel so strongly about it
5:33
in the plant pathology field… we’ve got t-shirts made that have this little phylogenetic tree here, and on the back, more interestingly (this is the way to remember it): “Bats are not birds, dolphins are not fish, oomycetes are not fungi”. So the fact that we’ve gone to the scale of making t-shirts about it tells you how strongly we feel about this important issue. But a lot of important pathogens are fungi. A way to think about what’s going on in a leaf
6:02
that has been colonised… It’s a bit like you’re on a petri dish. Microbes make these mycelia, the fungus makes these thin filaments, and the septa between them. This is a long, thin cell. You get branching… So you get this extensive colonisation of a petri dish here, with a fungus growing on it. And essentially, the similar kind of thing is going on within a leaf that results in enough nutrients being stolen from the host to give you spores that can then
6:36
propagate the disease to colonise another host. And the remarkable thing is if you’ve got this much stuff growing on you, how don’t you know it’s happening to you? So these are very stealthy pathogens. And they evade detection and also they suppress defence mechanisms. So what’s key for many of the filamentous eukaryotic pathogens such as fungi and oomycetes is the fact that you can elaborate these projections into the host cell called haustoria. And what these do is
7:20
they increase the surface area of intimate contact between the pathogen and the host, and there’s a tremendous set of transactions going across this boundary between them. There’s two membranes: there’s a host membrane and then there’s a pathogen membrane. So molecules are being pumped in by the pathogen to suppress defence, and sugars and so on are being sucked out. So these haustorial feeding structures called haustoria (single: haustorium) are key to this relationship.
7:54
And powdery mildews (and powdery mildews are fungi) also make these very elaborate… in fact, these are extraordinary. These are like little aliens within the cell. And then you’ve got tremendous increase in surface area with these finger-like projections at the end. And you get these white, fluffy sets of spores that are derived from the colonisation. The remarkable thing about this pathogen, the powdery mildews, is it only grows in the top layer of cells,
8:29
the epidermal cells. It doesn’t actually even get into the cells where all the sugar is (or
8:36
where the sugar’s being photosynthesised, I should say) but nevertheless, you get tremendous proliferation and these conidia then spread the pathogen to other uninfected plants. And there’s more of a cartoon of the haustorium. So again, I said there’s two membranes here: there’s the haustorial membrane and then there’s a space between them, and then there’s a host membrane around the outside. And topologically, this is not actually in the host cell. It’s within
9:14
the host cell wall, but it’s outside the host cell. But there’s a very extensive interface between the two, shown in more detail here. To get in, this pathogen and many others develop something called an appressorium, and this is basically a stiletto heel. Tremendous force is exerted that forces it through. Some of you may have heard Nick Talbot talk about this. You get hydrolysis and a big turgor pressure in this that forces the infection peg through into the host.
9:53
I’ve talked about downy mildews (in fact Phytophthora infestans, the late blight pathogen, is a downy mildew); I’ve talked about the powdery mildews, the ones that just colonise the cell’s surface; and the other major class of plant pathogen is the rests. So for example, here are three different species of wheat rust, leaf rust, stem rust and yellow rust, caused by pucciniomycete fungi, unlike the ascomycetes that cause the powdery mildews.
10:19
These also make haustoria, and these are the source of tremendous crop losses. And a theme that I will reiterate a bit is that wild relatives of our crops are actually a terrific source of new genes, additional genes for disease resistance. So wild eelops, brachypodium, et cetera… these wild grasses can be investigated for whether or not they carry genes that we can then use to improve our crops. Of course, because at least brachypodium
10:52
doesn’t form crosses with wheat, you’ll need to use the GM method to bring the resistance gene in. And here’s another example where the same method is being applied. Soybean rust is an interesting disease called Asian soybean rust, first defined in Japan. It’s been spreading across the world, it went and spread via Africa, and about fifteen years ago it blew in to Brazil. The spores blew across the Atlantic, and now it’s a two billion dollar problem on soybeans in Brazil, fostered
11:30
in part by the agricultural practices of you just, as soon as you harvest one lot of soybeans, you’re planting the next into where you just harvested. And we and colleagues have identified genes in a tropical legume called pigeon pea that confer resistance to Asian soybean rust. And if you get it out of pigeon pea, put it into soybeans, you’ve got a resistant soybean. So if you can go around
11:57
that loop a few times you could bring in a set of resistance genes that might give you durable resistance to this disease and avoid having to spray two billion dollars’ worth of fungicides. Now, not all plant pathogens make these haustoria. This is just one of a number of fungi which manage to just grow in the intercellular spaces of the leaf, the apoplastic extracellular spaces
12:26
of any leaf and the mesophyll, and the fungi just grow in these spaces. It’s not entirely clear the basis for their nutrition, but there’s enough sugars leaked into these spaces that they can then eventually emerge and sporulate and propagate their life cycles. So, I’ve talked about diseases that have a relatively intimate association with their host, and indeed it’s not in their interests to kill their host. But that’s not true of all plant
13:02
pathogens. Some plant pathogens are classified as necrotrophs and some as biotrophs. The biotrophs are the ones I’ve been telling you about; they need a living host to complete their life cycle. Whereas the necrotrophs will often make toxins, some of which are also poisonous to humans, that disable the defences and kill the host and enable the pathogen to feed
13:33
on what’s leaked out of the dead cells. I like to show this way of putting it: so the biotrophs are like blackmailers. The last thing you want if you’re a blackmailer making a nice living is for your source of money to die. Your biotrophs require these living hosts to complete their life cycle. I’ve shown you some haustorial pathogens. The alternative… the necrotrophs are cutthroats. They will eliminate the life of their target and grow on what’s left.
14:11
Botrytis cinerea is a classic necrotrophic pathogen: it kills its host and grows on the contents and sporulates rigorously. On this slide also is an Arabidopsis. This is a wild-type, non-mutant Arabidopsis that’s been infected with this necrotrophic pathogen, botrytis cinerea. And although if you look hard enough you can see a bit of activity and effectiveness of the pathogen, the plant is actually fighting off the pathogen reasonably effectively. But if you disable one
14:49
of the components of its hormone signalling involved in the ethylene response in Arabidopsis, you can see how disabled this plant is for immunity to this pathogen. And this makes another general point, which is that even if you’ve got an infected plant, it’s still mounting as much in the way of defence as it can. It’s not actually sufficient to completely thwart the pathogen, but the reason that you know that there’s a lot of defence going
15:17
on even in infected plants is you can find mutants where that type of defence has been disabled, and then those plants are even more susceptible. I mentioned this feature of toxins. So, this over here is a pathogen called Claviceps purpurea and it causes ergot of rye. In the Middle Ages, you’d sporadically have these so-called dancing sicknesses of whole villages, where, if the ergot had gotten into the grain, there’s a compound closely related to lysergic acid diethylamide,
16:00
which gives you a nasty surprise if you eat it in your loaves of bread and whole villages went nuts. But also, the compounds in this fungus can cause vasoconstriction and people lose limbs and so on.
16:21
Now, infections by pathogens and pests have all sorts of dramatic effects on their hosts. This, I think, used to be a carrot but it’s infested with root-knot nematodes. There’s plenty of examples of dramatic growth disturbances associated with infection, either by organisms like nematodes or by fungi. A couple of important concepts about insects… So there’s basically two kinds of insects that attack plants. There are those that eat stuff up,
16:55
like the Colorado beetle or the caterpillars that you might have seen on your cabbages. And then, the alternative is the suckers. You get aphids that stick a little stylet into the phloem cells and extract sugar without killing the host. This is essentially a more biotrophic insect than this. And I should say also that these insects… part of the problems they cause are through the transmission of viruses, which then go on to cause much more serious problems than the insect itself,
17:33
usually. A lot of important plant diseases are caused by viruses, and also by bacteria, in addition to the fungal diseases and the oomycete diseases that I mentioned earlier. So this is papaya, and this is papaya infected by papaya ringspot virus, and this is actually a resistant plant that has been engineered in a public sector program twenty or so years ago now to make small RNAs that target the virus. So all of the papaya that you eat that comes from Hawaii
18:10
is genetically modified to express small RNAs that prevent this virus from replicating on it. So when bacteria infect plants, where do they grow? If you’ve got a fluorescent protein label bacteria, you can see that its position corresponds to where you get legions that emerge as the disease develops, and the bacteria (rather like that fungus I mentioned earlier) grows in these intercellular spaces. There’s all sorts of biology on how the bacteria know when there’s
18:45
a high enough concentration of bacteria through quorum sensing, through detecting the presence of other bacteria, and then it switches on a whole program that enables it to overwhelm the host. Another very important bacterial disease that I’ll just go through because it’s one that plant biologists use all the time, at least the kind of plant biologists I talk to… The disease is caused by a bacteria, Agrobacterium tumefaciens, and it makes a disease called crown gall. And
19:21
why do you get uncontrolled growth of plant cells that have experienced agrobacterium? It’s because those plant cells have received DNA from the agrobacterium that perturbs hormone metabolism, usually through elevated expression of auxin and cytokinin. The agrobacterium has evolved a DNA delivery system that puts DNA into plant cells, that actually evolved from bacterial conjugation. So bacteria can move plasmas from one bacterial cell to another by a process called conjugation
19:59
– that’s how bacteria have sex. Basically, these bacteria have evolved to have sex with plant cells, and they deliver this DNA in the course of that and impose the crown galls on it. And what scientists have done is to identify the mechanism by which this DNA is delivered, the so-called virulence genes that define the transferred DNA, where it starts and stops, and package it up and get it across these two cell walls, the bacterial and the plant cell
20:35
wall (it’s still mysterious how it gets through the plant cell wall). And so, by taking advantage of these DNA delivery mechanisms but replacing the transferred DNA properties of hormone perturbation and replacing it with your favourite gene, you can put any gene you want into a plant cell, and then with various tricks you can select the plant cells that have received the DNA you want and have them grow and differentiate a whole plant. So this is just a little distant evolutionary
21:10
perspective on plants and animals and immunity. Plants evolve multicellularity independently of animals. The plant and animal taxa split (according to this phylogeny; I don’t know what the current favoured number is) but in 2002 the favourite number was about 1.6 billion years ago. So if you are a single-celled organism, your requirement for defence, or the kind of organisms that can grow on you, are very different than in a multicellular organism.
21:50
If you’re multicellular then you’ve got a lot of spaces between cells that bugs can get in, that you don’t have if you’re single-celled. They will have had to independently evolve resistance mechanisms, although there’s quite a lot of commonality in what they came up with.
22:09
Among the set of defences that plants have, they make reactive oxygen species like hydrogen peroxide or superoxide, as a dedicated enzyme to do this. Plants in defence against these pathogens can secrete antimicrobial peptides. They also can induce the biosynthesis of molecules that are antimicrobial; sometimes they’re called phytoalexins. And also (and you can imagine, this is a particularly useful mechanism against those pathogens that require a living host) they
22:47
can implement what’s called the hypersensitive cell death response, or hypersensitive response, or HR, as it’s often shortened to. So reactive oxygen species, antimicrobial peptides, antibiotics, cell death, and this is just a cell death response shown by these fluorescent compounds that accumulate as the cells die when this pathogen is trying to get into a resistant host. So how did this cell, under attack by this pathogen that’s trying to enter it, know to turn on all
23:26
these defences? I’ll be getting to that later. Here are a few more examples of this. This is if you’ve got a resistant Arabidopsis against a downy mildew. You can reveal where cell death is happening with trypan blue staining. Living cells exclude the dye, dead cells accumulate it, and so these are where cells are dead or dying where a pathogen is trying to get in. Tobacco that’s resistant to tobacco mosaic virus… all these legions here are due to
24:03
the virus starting to replicate, activating defence, the virus escaping to the next cell, defence activated… and eventually, the defence catches up with where the virus is and there is enough cell death for the virus to stop proliferating. And of course, viruses are almost the ultimate biotrophs, because they really need a living host to replicate. I mentioned reactive oxygen species. There’s a stain we can use to see reactive oxygen species;
24:35
it’s called diaminobenzidine. So, here we’ve got this diaminobenzidine staining where defence is being activated. We know some of the components that are required for this to happen: there are genes called respiratory burst oxidase homologs (nice little name), so we call them RBOH genes for short. And what these do is that they’re remarkable enzymes that have six transmembrane domains. They cover a cell membrane, and what they do is they take
25:15
electrons from this compound and it travels through this tube, basically, made by this protein. And these electrons then get dumped on oxygen to make superoxide, and superoxide then breaks down to make hydrogen peroxide. And if you get a mutant in this particular gene, RBOHD here, then you can see how the staining that you have in wild-type just goes away because you’ve lost the capacity for this process to be implemented. Now, I talked about detection mechanisms,
25:46
and detection mechanisms in the plant of pathogens are often encoded by what for historical reasons are called resistance genes. Resistance genes have been used by plant breeders, either through conscious or unconscious selection, from the earliest days of agriculture. They encode immune receptors that detect something from the pathogen, and then if they detect it, then defence is activated. But one of the frustrating things for plant
26:22
breeders is you can identify some new source of resistance, and you backcross it to let’s say your favourite wheat that’s got terrific yield several times, so that after enough backcrosses you’ve got that wonderful wheat but with a resistance gene in, and then you start planting it and it does really well. But then the pathogen evolves to overcome that resistance gene, and then the resistance gene doesn’t work, and then you need a replacement resistance gene. This
26:57
is called the boom and bust cycle. And actually, what we’re hopeful of (but this hasn’t ever been achieved yet, but I think we can see our way clear that we might be able to) is ways of breaking this boom-bust cycle and just staying up with the boom. That’s where we want to be. So, resistance genes encode immune receptors. There’s basically two kinds of immune receptors: there’s one that act at the cell surface and detect molecules outside the plant cell,
27:31
and there’s one that act within the cell that detect pathogen molecules that get into the cell. So I’m going to start off going through the cell surface receptors.
27:44
For a long time, people have been studying molecules and pathogens that seem to activate defence, and there was a lot of descriptive work that said, “Okay, this molecule, this glycan or whatever, turns on defence.” And people got stuck with that. People couldn’t get at the mechanism. But the key to getting at the mechanisms was being able to exploit genetics. This was first achieved with a particular molecule from pathogens that activate defence, and it’s called flagellin.
28:14
Bacteria have these flagella; it’s how they swim. These flagella are remarkable structures. They’re polymers of many, many copies of this protein, flagellin, and it forms a filament. So here’s a molecule of flagellin; it's one of the components of this set that make up the filament. Flagellin has within it a small peptide, this amino acid sequence. And basically, this is quite conserved across most bacteria, this peptide activates defence. So then, what was done
28:52
was to look for mutants that no longer responded to this and then to clone the gene, and what was identified was this gene called FLAGELLIN-SENSING 2. It recognises this flagellin peptide. It has a protein motif that is called a leucine-rich repeat. It’s a well-defined structure now, and it’s present in many proteins that interact with other proteins. And this leucine-rich repeat domain here has a transmembrane domain and then there’s a protein kinase domain inside,
29:27
and when you activate this, a bunch of magical things happen and defence responses are activated. And this is important, because it turns out that if you look at a mutant for an FLS2, it’s significantly more disease-susceptible (I think it’s this one here) compared to wild-type, and so this is genuinely contributing to the resistance of the plant. So maybe it’s seen more easily here; here’s an FLS2 mutant, and here’s a wild-type after it’s been inoculated
30:07
with pseudomonas, and you can see there’s a lot more symptoms because there’s more bacterial proliferation. And you can see the same sort of thing here compared to this wild-type. And in fact, there’s a lot of these cell surface receptors in plants that are important for immunity. Some of them sense this peptide from bacterial EF-Tu, an elongation factor involved in translation. Again, very well conserved across bacteria. And then there’s other
30:37
receptors that recognise chitin, which is a cell wall component of essentially all fungi. And this, again, is a molecule that the pathogen can’t help making. And then there’s a set of protein kinase activations that result in a defence response. So there’s more detail about that. This is now a bit of alphabet soup, which I probably don’t want to go through in detail, but there’s a lot of
31:12
components that are downstream of this activation by flagellin, have now been identified. And in particular, there’s a cell surface leucine-rich repeat kinase called BAT1 that’s absolutely required for this flagellin response, for this FLS2 to work. So upon flagellin provision, this molecule and this molecule, which were previously separate, come together and that induced proximity then is responsible for changes in the protein kinase activity of these bits inside,
31:47
and the chain of events that gives you defence. And what’s very interesting about this (and this is, I think, exciting from the standpoint of controlling plant diseases) is that you can move these recognition capacities from one plant to another. So different plants have different pattern cognition capacities, and these can be moved between species if you’ve identified the gene that confers recognition of a specific molecule. So, Arabidopsis, in fact all
32:21
the Brassicaceae, can recognise this EF-Tu via the EF-Tu receptor. It turns out that tomatoes, Solanaceae and in fact most other plants do not have the capacity to perceive this bacterial molecule. And if you take the Arabidopsis gene into a tomato, then you can confer resistance to bacterial diseases. So this is the disease that results from Ralstonia solanacearum, bacterial wilt. If you’ve put in the Arabidopsis EFR gene, then suddenly you’ve got elevated
33:01
resistance and essentially you’ve got no symptoms, at least in this experiment. So this has now got great potential for enhancing the recognition capacity of our crop plants and thus elevating resistance, because plants have got really great defence mechanisms. I went through them: active oxygen, programmed cell death, antimicrobial peptides, secondary products. And the way that a pathogen succeeds is to avoid the activation of these defence mechanisms,
33:34
either through evading perception or by shutting down those defence mechanisms. What this tells you is if you can increase the perception capacity of a tomato for bacteria, then its endogenous defence mechanisms will take care of reducing losses to that disease.
33:54
But nevertheless… I’ve told you about this great mechanism, pattern triggered immunity. You can detect these molecules that pathogens can’t help making like chitin and flagellin. Nevertheless, you get disease. So what’s the trick? The trick is to deliver proteins that are called effector proteins from the pathogen into the host. And bacterial pathogens, including pathogens of humans and other animals, have developed this secretion system called the
34:27
type 3 secretion system. It actually evolved from the mechanism to make flagella. What you can see here is these pili that are produced when you activate the type 3 secretion system for pseudomonas, and you can actually detect by immunolabelling in these experiments proteins such as AvrPto that have been delivered or are being delivered, in the electron microscope image here.
34:58
Bacteria deliver effectors through this type 3 secretion system. There’s a mechanism that elaborates this pilus, and the effectors can then actually get through this. Again, these pili have to also go through the plant cell wall somehow or other, and that’s really not understood. And when they get there, they somehow or other inactivate defence. I should say fungi and oomycetes also deliver effectors, and all we know about them really
35:29
is that they’ve got a signal peptide for getting out of the fungus or oomycete, and then somehow or other they get into the host cell, possibly across that intimate interface that is the haustorium. It’s still a very active area of research to understand how these proteins get in. And then, what do they do when they get there? These bacterial type 3 effectors at least, many of them are well defined. These ones here, this may look like alphabet soup,
36:04
but these are different effectors that have been shown to inactivate various components of the signalling proteins that participate in the chain of events between detection of the pathogen here and activation of immune responses here. So it’s been a very active area to identify the function of the bacterial effectors in particular, but we’re doing the same now with oomycete and fungal effectors, to try and understand what bit of the plant
36:35
defence they’re shutting down. But for a number of them, it is pretty clear where they’re hitting in this chain of events to compromise immunity. So pathogens are really brilliant at shutting down host defence. How come there are any plants left alive? Well, I’ll come back to that. I want to say a bit about other effectors. Not all effectors suppress immunity. Some of them do something more subtle. This is a very interesting story: the effectors that are recognised by some
37:10
plant resistance genes carry a set of repeats of a particular 34 amino acid motif. They’re almost identical repeats except for amino acids 12 and 13 of these repeats. So they’ll go HD or NG, NS, NG, et cetera. And eventually it was realised that these are DNA binding proteins and these motifs here specify which of the nucleotides in DNA the effector binds to. So if you’ve got an HD here, it binds an A, an NG here it binds a T, and so on and so forth. And
37:51
there’s a crystal structure now of this effector that’s shown wrapped around DNA here. I mean, it’s a spectacular structure. If you look down the DNA molecule, you can see a pinwheel-type effect. And what these do is they turn on genes in the plant that render the plant more susceptible. So these are transcription factors injected from Xanthomonas by type 3 secretion, and what do they do? They go in and they make the plant more susceptible by inducing the
38:22
expression of proteins such as these proteins, the sweet proteins. What are sweet proteins? They are sugar transporters. So basically, what the pathogen can do is to impose on the host elevated expression of sugar transporters that pump sugar out so the pathogen can grow. In an additional twist, you can actually now engineer these so-called TAL effectors (transcription activator-like effectors) such that you attach them to nuclease domain
38:55
so they cut DNA, and you can drive them so they cut DNA within the promoter of one of these sweet genes. The results from these cuts is you get deletions in the promoter, and then you can end up with a plant that’s more resistant because the plant has lost the capacity to be activatable by that particular TAL effector. Right. So pathogens make all these effectors. The role of the effectors is to shut down host defence, or they render the plant more amenable
39:33
to being colonised by the pathogen. So how come plants aren’t wiped out? That’s my little segue from two slides ago. And that’s because plants have evolved the capacity to recognise effectors as a trigger to activate defence. So this is the second layer of plant immunity, and it’s often called effector-triggered immunity or ETI. Plants carry these proteins, nucleotide binding leucine-rich repeat proteins. They bind ATP or ADP in this nucleotide binding domain
40:09
here. They’ve got leucine-rich repeats in their C-terminus, and that region is almost certainly involved in the recognition of the molecule from the pathogen. And then they come in two main different flavours of signalling domain at their N-termini, either a tier domain or a coil domain.
40:29
So these resistance genes can be very effective. This is a GM field trial we did in 2012, I think, this one was. We’d cloned a resistance gene that encoded one of these NLR-type receptors from a wild relative of potato called Solanum venturii, put it into Maris Piper and lo and behold, we’ve got a resistant plant. This is now a field trial, as I say, natural infection. Whereas the that had not been transformed… well, you can see what happened to them. There’s not much left of them.
41:10
But I told you about the boom and bust cycle. If you just have one resistance gene in there, you’re actually making it relatively easy for the pathogen to make a mutation in the recognised component, and then overcome this resistance gene. And basically, there’s no magic wand to wave over this, but we think about it probabilistically – if you can clone different resistance genes and put them in the same plant in a so-called stack, then instead of the pathogen having to mutate to
41:44
overcome one resistance gene to grow, it’s got to make three simultaneous mutations in three different recognised effectors, and that stack should therefore be more durable. And we’re doing a field trial with these plants this year and next year and the year after, so we’ll see how it goes. Now I mentioned this boom and bust cycle. If resistance genes only last three years… you know, if they’re that useless, how come they stick around? I mean, why are we still lumbered with
42:16
these useless genes? And the answer to that is in fact that in plant populations, there’s normally tremendous genetic heterogeneity. If you look at a field of wheat out there as you’re driving here, you’ve got millions of plants that are genetically identical. And that’s represented on the left here as plants where you’ve just got the one red resistance gene; they’ve all got the same resistance gene. Here, you’ve got five different resistance genes that recognise five different
42:49
effectors, so you’ve got a mixed population and you’re going to have very different consequences for any epidemic. So let’s say this plant here develops disease… you know it’s going to end badly, and lo and behold, this field is toast very quickly. And of course, that’s what would happen in our crops were it not for fungicides. But if you’ve got a plant here that’s infected, then quite apart from anything else, it’s further epidemiologically to the next susceptible host.
43:22
So the whole epidemic of that particular race of the pathogen on this mixed population of plants is going to be slower. So they are effective, but any particular resistance gene is amenable to being overcome by mutations in the pathogen. So wild plants, especially in out-breeders… you’re going to have a lot of diversity in pathogen recognition capacity, as in the human major histocompatibility complex. In plants, you’ve got tens to hundreds of resistance gene loci,
43:56
many of which come in sets of alleles with different recognition capacities. This diversity in natural populations is key to effectiveness of resistance, because each pathogen strain is going to be likely to be poorly adapted to most of the individuals in the field. So at the very least, you’re going to have less of an epidemic. But what we do have in wild populations is a rich source of new resistance gene alleles that could help crop disease resistance.
44:28
And now we’re faced with a tactical question: how do we game evolution here? What’s the least bad way to game evolution? Of course, it’s like antibiotics. Antibiotics are great: you know, your kid needs antibiotics, you treat them with antibiotics, but if everybody does that with one antibiotic then the antibiotic loses effectiveness. So you need to manage any resistance mode of action you’re working with to maximise its durability. And of course, I showed
44:58
you the wonders of having a mixed population genetically of resistance recognition capacities, but that’s not much good for seed companies who want to sell bags of seed where all the individual plants in there are the same. So I think we’re left with stacking as the best way to improve on the boom and bust cycle that previous plant breeders have wrestled with. So what are these resistance genes? What we’ve got here is more alphabet soup, but the point of this
45:27
is that both plants and animals have NLR-type proteins. They’ve all got this ATPase domain, variously called a NACHT or embark, and the plants are just the embark domain, various signalling domains at the N-terminus, different sets of signalling domains at the N-termini of these animal genes and various other motifs too. I just want to mention this one that’s called a caspase recruitment domain, and highlight the NAIPs and NLRC4. Because what’s been shown is
45:59
in terms of the mechanism of those two proteins, NLRC4 is a downstream signalling component that has these NACHT domains and the caspase recruitment domain. It’s normally auto-inhibited, but when the NAIP detects molecules from pathogens (one of them is actually flagellin) it has a confirmational change, which means it activates the NLRC4, and then once it’s activated it interacts with other NLRC4s and you get this chain reaction essentially, where you have 11 or
46:37
so NLRC4s in this beautiful configuration, which has been studied by cryo electron microscopy, and the structure defined. And the result of this is you start with a monomer, with a caspase recruitment domain like this… nothing’s going to happen. Because you need multiple copies of this protein domain to recruit caspases, which then hydrolyse proteins and hydrolyse themselves and send off a chain of events. But this caspase recruitment domain is at the N-terminus of this,
47:10
and here you’ve got something that’s chock-a-block with caspase recruitment domains that recruits caspases, and then something happens. So we’re guessing that something similar happens in plants for plant resistance proteins, plant NLRs. We can draw cartoons that look like this, so this will be the off form, coiled-coil or tier, embark domain, leucine-rich repeats… there’s a molecule and it recognises an effector, it changes confirmation and something happens.
47:43
But actually how plant NLR proteins work is a complete mystery, which is one of the reasons I still get up in the morning, because I’m quite excited about trying to figure out how they work.
47:56
Now, something I do need to tell you is that these NLR proteins can either recognise a pathogen directly… so here’s a resistance protein, recognise an effector, stuff happens. Or I’ve told you that these effectors recognise or interact with molecules that participate in plant defence, and actually we know now that some of the resistance proteins are monitoring the state of these targets of effectors from pathogens, and they are
48:28
only activated when they detect a change in the virulence target that is effector-dependent. So either the effector is recognised directly by the resistance protein, or indirectly because the resistance protein is monitoring the effects of the effector rather than the effector itself.
48:46
I work with these two proteins which work as a pair, RPS4 and RRS1, the tier class, and the interesting thing about this class is that at the C-terminus, it has a protein that is not normally seen in a resistance protein. It’s a DNA binding domain of a working transcription factor family. Working transcription factors are transcription factors that play a key role in activation of plant defence gene transcription. Mutants in WRKYs are more disease-susceptible.
49:20
So why on earth is this WRKY domain sitting in the middle of a resistance protein, or near the end of a resistance protein? These two genes, RPS4 and RRS1, they’re right next door to each other in the genome, and they’re each defined by the effector that they recognise. For this one to work it needs this one, and for this one to work it needs this one. So they absolutely have to work together.
49:46
And what we now know is that they form a complex like this, and what’s going on in this complex is that when effectors show up with these names whose job it is for the pathogen to interfere with the function of WRKY transcription factors, so for example popB2 acetylates the DNA binding domain of its WRKY transcription factor so it can no longer bind DNA. It just doesn’t work anymore. So the plant has evolved these complex receptors that enable it to know when
50:22
a pathogen has showed up that is messing with WRKY transcription factors, because the action of this thing on WRKYs… because that’s what it does, it can’t avoid acting on this WRKY domain that’s in the resistance protein, and the changes that ensue upon that interaction activate defence and you get resistance. So it’s very cute. It’s like judo, you know, you use your opponent’s attack as your defence. And it turns out there are a lot of proteins
50:51
like this. So RRS1 and RPS4 I’ve just mentioned, there’s this WRKY domain here. Here’s a pair from rice, that recognise effectors from rice blast in this domain. And there’s a nice analysis of this; it’s called the integrated decoy hypothesis. So this WRKY domain acts as a decoy that enables the action of effectors to be drawn into action on something that when that action happens, defence is then activated. And this is pretty interesting because these R gene
51:27
pairs like this are very good at working when you move them from one plant species to another. And so, something I’m really excited about at the moment is this grant that I have called ImmunityByPairDesign, and what we want to do is to understand the design principles of this complex and then also, if we figure them out well enough, to design in the capacity to respond to different effector actions. So can we put some other domains in where the WRKY domain is and build a complex
51:59
that enables to detect the action different kinds of effectors that hit different kinds of domains. So just to sum up, there’s various representations of this. There’s two different kinds of representation here, so maybe I’ll start off with this cartoon. You’ve got fungi or oomycetes and they make molecules they can’t help making, that are detected by cell surface receptors that activate defence. Bacteria and fungi pump in these proteins called effectors, whose job it is
52:37
to interfere with this, but the plant has evolved the capacity to detect some of these effectors and the result is effector-triggered immunity. I like to represent it… it’s a bit like the Z scheme for photosynthesis. So, pattern-triggered immunity activates defence (that’s your PTI) but effectors trigger susceptibility, so this cloud of effectors here can drag down the level of activation of defence so then you’ve got susceptibility, but then if one of them’s
53:06
recognised by a resistance protein then you’ve got elevated defence, a higher amplitude of defence, and that gives you resistance. And this is not just fungi, oomycetes and bacteria, because it’s clear now that nematodes and aphids also make effectors whose job it is to interfere with host defence mechanisms. So this is another cartoon of it. I work at the Sainsbury Laboratory in Norwich, and we have a great set of colleagues. Susan and Zilka work on
53:45
the cell surface receptors, Sophien on effectors and also on resistance proteins, and this is Matt and Peter. And this is my wonderful team at the Sainsbury Lab at the Christmas event. And I’d like to thank these entities for funding, particularly Gatsby, obviously. Thank you.
Here to help you learn
Sign in to keep a private conversation with your course tutor.
Ask me anything about the material…
Sign in to chat