Session 9: Coevolution
Transcript of Part 4: Nicotiana attenuata’s Responses to Attack from a Nicotine-tolerant Herbivore
00:00:13.05 My name is Ian Baldwin and I'm delighted, here, to be presenting Part 2 in a three-part 00:00:18.15 story on how to study the plant ecological interactions in the genomics era. 00:00:24.18 I'm a Director of the Max Planck Institute for Chemical Ecology. 00:00:28.16 And in Part 2, here, I'll be talking about Nicotiana attenuata, the plant that is right 00:00:35.12 here, it's ability to be able to respond to attack from a nicotine-tolerant herbivore. 00:00:42.12 And I just want to remind you this is Part 2 of a three-part series and in the third 00:00:48.04 part I'll be talking about the plant's perspective on sex, seeds, and microbes. 00:00:54.24 In Part 1, I talked about how the Max Planck Institute for Chemical Ecology came about 00:01:03.01 and how it fits into the rich history of the field of plant-herbivore interactions, and 00:01:06.24 how Ernst Stahl, in 1888, really started the field. 00:01:11.19 I also talked about the process of training genome enabled field biologists and how to... 00:01:18.05 how they are trying to phytomorphize themselves and understand what plants are doing with 00:01:23.00 this incredible chemical prowess that they have, and how they use those chemicals to 00:01:28.10 solve ecological problems. 00:01:30.09 I also introduced the "ask the ecosystem" approach, which combines both field and laboratory 00:01:36.02 studies of transgenic plants, and introduced the important process of silencing genes to 00:01:43.15 understand their function at a Darwinian level in an organismic context. 00:01:55.05 These field experiments are conducted with these genetically modified plants in their 00:01:59.16 native habitat in a nature preserve in the southwestern deserts of the United States, 00:02:05.14 in Utah, in a collaboration with Brigham Young University. 00:02:10.04 What I want to do here in Part 2 is talk about this particular interaction that's unfolding 00:02:15.24 to you right here. 00:02:17.22 This is an interaction of the plant that we work, Nicotiana attenuata, and the hawk moth, 00:02:25.24 Manduca sexta and Manduca quinquemaculata. 00:02:28.23 It's a remarkable interaction filmed here in fast motion, fortunately enough, by the 00:02:35.03 team of Volker Arzt from the movie Kluge Pflanzen, and they were so kind for letting us use their 00:02:40.09 outtakes. 00:02:41.09 This is a remarkable interaction because the plant is chock-a-block full of one of the 00:02:47.01 most toxic compounds for human beings, and for almost any animal with a neuromuscular 00:02:52.17 junction, namely, nicotine. 00:02:54.11 Now, many of us have had an addictive relationship with nicotine as smokers, but if any smoker 00:03:01.23 had ever tried to eat a Nicotiana plant you'd realize just how poisonous this plant is. 00:03:08.21 Nicotine poisons the neuromuscular junction, the acetylcholine receptor called the nicotinic 00:03:15.07 acetylcholine receptor, and that receptor mediates how muscles move. 00:03:21.03 Now, if you were a plant and you wanted to design a chemical defense which would poison 00:03:28.09 animals that moved with muscles, this would be an ideal defense compound to produce. 00:03:33.17 And this is exactly what Nicotiana attenuata and some of the other tobacco plants have 00:03:38.08 done -- they've evolved this molecule. 00:03:40.07 Now, this molecule evolved from two primary metabolic pathways, the NAD pathway and the 00:03:45.21 polyamine pathway, that produced the two rings that both contain a nitrogen and then they're 00:03:50.16 fused together to form the molecule... the molecule nicotine. 00:03:55.11 Nicotine is synthesized, as I said, from these two primary pathways, and its biosynthesis 00:04:00.02 has been worked out by a number of researchers over time. 00:04:03.07 But what is more recent is its understanding of the evolutionary history of this biosynthetic 00:04:08.08 pathway. 00:04:09.08 And this was done recently by Shuqing Xu in our department and a number of his colleagues 00:04:15.17 in the informatics group that is involved in assembling the genome of Nicotiana attenuata, 00:04:21.03 which is currently under review. 00:04:22.13 And what Shuqing Xu and colleagues found out was that umm... all of the genes that are 00:04:28.02 involved in nicotine biosynthesis are genes that are part of a whole genome triplication 00:04:35.13 event that happened with the Solanaceae, namely, all the plants that are of the group of plants 00:04:42.03 that are called solanaceous plants: potatoes, tomatoes, eggplant. 00:04:48.06 All went through a genome triplication event. 00:04:50.22 Those extra copies of the genes were therefore given the evolutionary privilege to be able 00:04:56.02 to be combined in novel things other than their primary metabolic pathways. 00:05:00.09 And potatoes and tomatoes and tobacco all produced nicotine, but tomatoes and potatoes 00:05:06.24 produce them at much lower levels -- about three orders of magnitude lower than tobacco 00:05:11.12 plants. 00:05:12.20 Tobacco plants' remarkable ability to produce enormous quantities of nicotine, to really 00:05:17.21 make it defensive, and smokeable, has to do with the ability of the plant to have corralled 00:05:25.11 the biosynthesis of those pathways into the roots, and to have fused the two rings in 00:05:30.16 a very efficient way, and funnel a lot of reduced nitrogen into the biosynthetic pathway. 00:05:36.08 That's described in this paper that is currently under review. 00:05:39.24 Now, nicotine biosynthesis can be inhibited by silencing a single gene. 00:05:45.14 This gene, here, putrescine methyltransferase, which we have silenced by RNAi and been able 00:05:51.13 to produce plants that are relatively nicotine-free. 00:05:55.01 And when you make a plant that's relatively nicotine-free and you take it back out into 00:05:59.06 the native habitat and plant it in some natural habitats, you realize just how effective this 00:06:04.00 defense is. 00:06:05.00 Because every deer, every rabbit, every gopher in the neighborhood finds out about it, and 00:06:11.11 here's an example of a gopher that's coming up, has dug a special tunnel up underneath 00:06:16.13 this nicotine-free plant and is pulling it down to its burrows. 00:06:20.00 So, without nicotine, the plants become quite defenseless and are stripped bare of their... 00:06:27.13 of their phloem by rabbits and other mammal... mammalian browsers them, and usually don't 00:06:32.17 last very long. 00:06:34.16 Now, Manduca sexta, which was gobbling, devouring those plants in that first video that I showed 00:06:39.21 you, is able to do it because it is... well, it basically holds the world's record for 00:06:45.14 nicotine tolerance. 00:06:47.00 If you compare the LD50 -- the lethal dose at which 50% of an experimental population 00:06:52.15 dies -- you realize that even the most hardcore, [unknown], carton-a-day smoking human being 00:07:00.23 still has an LD50 that is 750 times lower than that of Manduca sexta, which is about 00:07:08.18 1500 milligrams per kilogram that it's able to tolerate. 00:07:12.11 Now, it's been known since the '60s that Manduca sexta's tolerance of nicotine is based on 00:07:19.11 a physiology that allows it to excrete all the nicotine that it ingests without any apparent 00:07:26.04 metabolism at... or any apparent effect on its nervous system. 00:07:30.22 How it does that is still very much an active area of discovery, but, as we look at a caterpillar 00:07:38.05 eating a plant, we've been interested in asking the caterpillar, transcriptomically, what 00:07:44.10 is it doing inside of its gut to be able to handle those many human doses of lethal doses 00:07:50.22 of nicotine that it's ingesting almost on an hourly basis. 00:07:55.00 And when you ask the caterpillar, transcriptomically, there is consistently one cytochrome P450 00:08:02.09 which is constantly being regulated in direct proportion to the amount of nicotine that 00:08:06.00 is ingested by the caterpillar. 00:08:07.07 And this is a cytochrome P450 with a long complicated name called 6B46. 00:08:14.11 And you can see it regulates at a high level when it's eating nicotine-containing plant 00:08:18.15 and downregulates when it's eating nicotine-free plants. 00:08:22.02 So, to understand what this particular gene was doing in the caterpillar and why it was 00:08:28.04 being up-regulated every time the caterpillar ate a high-nicotine-containing plant, two 00:08:33.13 scientists in the department, Pavan Kuma and Sagar Pandi, designed a procedure that allowed 00:08:44.06 the study of this particular gene to occur in the natural environment of both the insect 00:08:48.21 and the plant. 00:08:50.03 And what they did was they took that gene, made a double-stranded contract, which is 00:08:53.14 depicted here in yellow in the plant, transferred it into the plant so that was consistently 00:08:58.10 expressing this double-stranded piece of the gene that they wanted to silence, and then 00:09:03.23 they planted it out in Utah and let free-ranging caterpillars feed on them. 00:09:09.16 And, in that process of feeding on these particular plants, the caterpillar ingests double-stranded 00:09:15.04 and then the gene in the caterpillar gets silenced. 00:09:18.19 And in those genes silence caterpillars they were able to understand the function of that 00:09:24.22 particular cytochrome P450, which is up-regulated during the defense process. 00:09:31.08 And what they discovered was really remarkable, but let me show... first show you some data 00:09:35.04 on just how effective this plant-mediated RNAi process is. 00:09:40.08 Here on the y axis is the transcript levels for the particular gene that they're looking 00:09:44.21 at in the various tissues of the caterpillar. 00:09:47.15 And I want you to focus particularly on the midgut, which shows that caterpillars eating 00:09:54.18 nicotine-containing plants have very high levels of that transcript. 00:09:58.14 But if the caterpillars are feeding on a nicotine-free plant, the transcript levels are quite low. 00:10:04.07 But if the caterpillars are feeding on one of these PMRi... 00:10:08.03 PMRi plants that are expressing a double-stranded construct of that cytochrome P450, and those 00:10:14.15 plants contain normal high levels of nicotine, you would expect the transcript levels to 00:10:19.16 be this high, but instead they're that low. 00:10:22.21 And they're that low because the gene is being silenced by that plant... by the plant's food, 00:10:31.01 and the caterpillars are ingesting that gene and that RNAi process is happening in basically 00:10:36.19 free-living, free-ranging caterpillars in the field. 00:10:39.15 It's a remarkable experimental tool that allows us to study plant-insect interactions in nature 00:10:45.04 using genetic tools to manipulate not just the plant, but also the insects that are feeding 00:10:50.01 on the plant. 00:10:51.04 Now, what's remarkable about this story is that it was actually a wolf spider occurring 00:10:56.04 in the natural habitat of the plant that told us the function of this particular gene in 00:11:03.12 the caterpillar. 00:11:05.09 And now I'm going to show you a series of videos and here's a video of a wolf spider 00:11:09.14 attacking a nicotine-free plant and you can see from that video that it just gobbled it 00:11:14.21 up. 00:11:15.21 So, if the caterpillar is feeding on a nicotine-free plant it has no nicotine in it and the wolf 00:11:21.05 spider finds it as food. 00:11:23.21 Now, here is, in the next video, a spider attacking a caterpillar that is fed on one 00:11:30.01 of these PMRi plants. 00:11:31.14 Now, remember those have are full of nicotine but they are silencing this particular gene 00:11:36.17 in the caterpillar. 00:11:37.18 And you can see from this video that the caterpillar is attacked and eaten as if it was nicotine-free, 00:11:44.06 and this was discovered by the two scientists who had placed caterpillars on plants out 00:11:51.04 in the field, having them feeding on these particular plants that silence the gene in 00:11:55.05 the caterpillar, and all the caterpillars disappeared at night. 00:11:58.12 And the wolf spider hunts at nighttime and that's how they found the wolf spider. 00:12:02.18 Now, here is the key moment, the key observation that allowed them to understand what was going 00:12:08.09 on, because in the next video here is a spider attacking a nicotine-containing plant, that's 00:12:17.01 a normal empty vector wild-type plant, and you can see all it did was go up and palpitate 00:12:22.21 the spider... the caterpillar and then it immediately backed away. 00:12:26.06 And what was going on in that palpitation, that little moment when the caterpillar being 00:12:31.07 assessed by the spider and the spider decided, oh... 00:12:33.15 I'm not gonna eat this, was that the caterpillar was, through its spiracles... caterpillars 00:12:40.16 have 17 spiracles, they are basically the lungs of the caterpillar... caterpillars have 00:12:46.06 all these tubes and that's how they exchange air... and through the spiracle the caterpillar 00:12:51.01 is puffing out a load of nicotine into the face of the caterpillar... into the face of 00:12:57.06 the attacking spider. 00:12:58.06 And that's why the attacking spider jumped away. 00:13:01.11 And what this gene is doing is mediating that process, in a way that we don't really understand 00:13:06.08 biochemically, allowing the caterpillar to basically divert a lot of... some portion 00:13:11.13 of that massive amount of nicotine that's flowing through its gut, that it's excreting 00:13:15.02 out, but then it moves it into the spiracles and it uses it defensively when a spider comes 00:13:20.03 up and says, are you good food?, and the caterpillar then just puffs out this thing of nicotine 00:13:23.23 and repels it, okay? 00:13:26.10 So, that shows you that, actually, the caterpillar, even though it's excreting most of its nicotine, 00:13:32.16 is using it defensively, it's co-opting just a small fraction of what's going through its 00:13:36.19 gut for its own defensive purposes. 00:13:38.09 But, now, what I'm going to tell you, for the rest of this talk, is what happens when 00:13:43.20 the plant recognizes that it's being attacked by that particular nicotine-tolerant caterpillar. 00:13:50.21 Because that recognition process results in six changes in the plant that all involve 00:13:58.07 how the plant deals with a caterpillar that has broken through one of its major defenses 00:14:04.00 and has to figure out something else to do with this guy that's going to eat it, and 00:14:08.18 that's going to make lunch of it. 00:14:10.08 And that recognition process starts right here. 00:14:13.15 And if you look right at that cut leaf edge, there, you can see a little bit of green slimy 00:14:18.10 stuff that the caterpillar is leaving on the edge of the leaf. 00:14:21.19 Now, it turns out it's not doing that intentionally, that's just part of the eating process, it's 00:14:25.13 part of its oral secretions, that's part of the process of masticating the leaves to be 00:14:29.06 able to digest it, but in those oral secretions are a group of compounds that are called fatty 00:14:35.07 acid amino acid conjugates. 00:14:36.24 FACs is what we call them, and the structures of those FACs are right here. 00:14:41.11 They're very simple molecules -- they're just fatty acids esterified to amino acids. 00:14:45.16 There's two of them, there's five fatty acids, and they make basically eight different structures, 00:14:50.04 and those eight structures are what the plant uses to say, aha I'm being attacked by Manduca 00:14:57.24 sexta and I know that it's nicotine resistant in some way or another. 00:15:00.23 And those are... 00:15:01.23 I'm anthropomorphizing but that's basically the message. 00:15:04.20 Now, what I'm going to do... this is, by the way... this fatty acid amino acid conjugates 00:15:09.06 were discovered by Rayko Halitschke in his thesis and published back in 2001. 00:15:13.14 What I'm going to do now is to take you through those six layers of defense, avoidance, and 00:15:19.24 tolerance that the plant goes through when it recognizes this... that it's being attacked 00:15:27.01 by this... this particular caterpillar. 00:15:31.00 And those six layers are both an up and down regulation of direct defenses, a bunch of 00:15:35.23 indirect defenses, an interaction between indirect and direct defenses, tolerance responses, 00:15:41.07 and avoidance responses. 00:15:43.02 So, follow with me and we're going to go through this remarkable journal... journey of what 00:15:47.23 happens to the plant as it reorganizes its metabolism, physiology, to deal with the fact 00:15:54.19 that it's got a predator that it really has to deal with. 00:15:58.11 Okay. 00:15:59.11 Now, first I want to talk a little bit about the recognition process. 00:16:02.15 So, umm... we've been able to, because we have these synthetic fatty acid amino acid 00:16:08.23 conjugates, we have the elicitors... we're able to start the interaction between plant 00:16:14.05 and its responses without having to have a caterpillar. 00:16:17.06 So, we simply just take a pattern wheel and we add these oral secretions to spit to the 00:16:21.10 leaves, to the holes that are made in leaves with the pattern wheel, and that elicits a 00:16:25.05 very complicated set of signaling responses. 00:16:28.14 We haven't identified the elicitor... the... the receptor yet for the elicitor. 00:16:32.03 We know the elicitor -- those are the FACs, the receptor is unknown, but that that elicits 00:16:38.12 a very complicated signaling network that involves MAP kinases, SIP and WIP kinases, 00:16:43.19 the jasmonate signaling cascade, and a lot of modulation of that jasmonate signaling 00:16:50.11 cascade through other kinases, the activation of CDP kinases as well, and the perception 00:16:59.04 by other receptors, LecRK, that it basically involves a regulation of jasmonate signaling. 00:17:06.05 And, because caterpillars do not brush their mandibles when they eat a plant, they also 00:17:11.14 contain bacteria and other sorts of bacterial signals, and the plant has to make sure that 00:17:16.18 it's activating a jasmonate signaling cascade and not a salicylate signaling cascade, so 00:17:22.17 all of this signaling has to do with being able to make sure that the caterpillar doesn't 00:17:28.09 fake out the plant with its bacterial signals, but rather generates a nice clean jasmonate 00:17:35.07 response, which activates five out of the six layers that I'm now going to talk to you 00:17:39.10 about. 00:17:41.03 Now, that was a lot of work, and that work was done by some remarkable group leaders 00:17:48.00 and a remarkable number of talented students that I wish I could talk about in greater 00:17:52.16 detail -- but here are their pictures. 00:17:55.13 It also illustrates another important message that I want to bring up in this talk and that 00:17:59.17 is that interplay between mechanism and function, that if you understand the details by which 00:18:06.00 these responses come about, you have the very tools that you can manipulate genetically 00:18:11.13 to be able to create plants that are not able to show the response, and all of those steps 00:18:17.00 in those signaling pathways have been very useful tools to allow us to be able to manipulate 00:18:22.23 some aspects of these six responses in different combinations, and test them functionally in 00:18:27.15 the field, in the actual habitat in which the plant evolved. 00:18:31.23 Now, let me go through the six responses. 00:18:34.00 The first response was the up- and down-regulation of these, what we call, direct defenses. 00:18:39.07 Now, direct defenses basically can be categorized in two groups. 00:18:43.05 They're either toxins, things that poison animals that eat plants, without poisoning 00:18:49.10 the plant too much, and are specifically targeted against the things that are different between 00:18:54.15 animals and plants, like nervous systems; plants don't have a nervous system, so it's 00:18:58.19 really easy for plants to make nervous system poisons that are not toxic to them, but are 00:19:04.18 very toxic to the animals that want to eat them. 00:19:07.01 So, in addition to toxins, there's also another type of direct defense that are called digestibility 00:19:12.08 reducers. 00:19:13.08 They're basically interfering with the main reason why a caterpillar wants to eat a plant 00:19:18.11 in the first place, which is to turn caterpillar protein... plant protein into caterpillar 00:19:23.23 protein, to turn caterpillar... plant energy substances like glucose and sucrose and starch 00:19:30.02 into energy substances that the caterpillar can use. 00:19:33.11 So, that digestibility process can be interfered with lots of different ways. 00:19:40.00 Interfering with all the steps of ingestion and digestion... for example, there are protease 00:19:45.06 inhibitors we're going to talk a little bit about, there are tannins and amylase inhibitors 00:19:48.11 that are basically affecting the digestive enzymes that take apart plant proteins and 00:19:52.21 starches, and make them available to be uptake... taken up by the guts of caterpillars. 00:19:57.24 But there's also abrasives, things that wear down the mandibles and the teeth of the herbivores, 00:20:02.20 because, you know, if an herbivore doesn't have a pair of teeth, a set of mandibles or 00:20:08.18 a set of teeth, it can't chew a plant. 00:20:10.24 And plants fill themselves with silica and other sorts abrasives that just wear down 00:20:15.18 the teeth. 00:20:16.18 And there is no easier way to starve an ungulate than to wear out its teeth, and plants do 00:20:22.17 that all the time. 00:20:23.17 Now, I just want to talk about the down-regulation, as well as the up-regulation, because the 00:20:28.09 first thing that happens when those FACs are recognized by the plant is the plant has an 00:20:34.02 ethylene burst and shuts down the very gene that we silenced to make a nicotine-free plant. 00:20:38.24 And that's in fact the reason why we did it, because we learned from the caterpillar how 00:20:43.17 it was shutting down nicotine biosynthesis in the plant. 00:20:48.02 And it's very clear, now, that since the caterpillar is co-opting a certain portion of the nicotine 00:20:53.13 for its own defense, the plant is most likely down regulating its nicotine production so 00:20:59.00 that the plant... so the caterpillar can't co-opt the extra nicotine it produces. 00:21:02.23 If it was a deer or a rabbit producing... doing the damage rather than a Manduca sexta 00:21:08.14 larvae, nicotine production would be operated 5- or 6-fold and the... you know, the plant 00:21:14.17 would become even more full of nicotine than it already is, so that a single leaf would 00:21:20.00 have the same amount of nicotine in it as half a carton of [unknown] cigarettes. 00:21:24.13 So, that massive up-regulation process is just basically stopped and the... the plant 00:21:30.01 is down-regulating nicotine production when it knows that it's being attacked by a nicotine-resistant 00:21:34.10 caterpillar. 00:21:35.10 Okay. 00:21:36.10 Then it produces a whole bunch of other types of compounds, many of which we had no idea 00:21:41.06 what they did. 00:21:42.06 And I just want to talk, just briefly, about a group of compounds called diterpene glycosides. 00:21:47.03 This is some work done by a PhD student who's just finishing enough, Sven Heiling, and he's 00:21:51.06 done some beautiful analytical work characterizing these molecules that were basically unknown. 00:21:56.15 There were 46 of them in Nicotiana attenuata and they are basically produced in the chloroplast 00:22:05.23 by what's called the MEP pathway and the DOX pathway, to produce a basic backbone diterpene 00:22:13.02 structure, and that backbone diterpene structure is depicted there. 00:22:17.10 It's hydroxylated and then sent out to the plant and decorated further by enzymes that 00:22:24.11 add different types of sugars to them -- I'll talk about that a little bit later. 00:22:29.12 But, because this is a secondary metabolic pathway, the main enzyme that's involved there, 00:22:35.22 this NaGGPPS that is highlighted in bold, there, also has three copies, because of that 00:22:43.12 trip... the genome duplication event, and, if you silence the one that's dedicated for 00:22:48.04 the production of these pathways, you can completely take out the whole biosynthetic 00:22:52.03 pathway by one gene silencing step. 00:22:54.20 So, by silencing that particular gene, we're able to make DTG-free plants and if you fed 00:23:01.07 them to caterpillars you can see that the caterpillars basically were able to increase 00:23:06.19 their growth rate almost fivefold when they're feeding on these DTG-free plants. 00:23:11.19 So, even though we had no idea that these were toxic or defensive when we looked at 00:23:17.07 the structures and figured out their structures, when we silence them and produce plants that 00:23:21.05 were where DTG-free the caterpillars told us that, oh... this is really a pretty nasty 00:23:26.17 defense compound. 00:23:28.18 And what Sven has been able to do is to identify all the different enzymes that are involved 00:23:33.22 in decorating them with sugars of various sorts, glucose and rhamnose, here, and then 00:23:40.18 they are malonated in addition, and that's what generates all those 48 different... 00:23:43.19 48 different structures. 00:23:44.20 Now, it turns out that if you look at the... the poop of a caterpillar, the frass that 00:23:50.04 comes out the busi... the other end of the caterpillar after it's eating leaves, Spoorthi 00:23:54.15 Poreddy, who is a PhD student who just finished up, along with Sven and Jianciai Li, have 00:23:59.21 been discovering that there's a very interesting dynamic that's going on in the caterpillar's 00:24:06.13 gut as it's trying to remove particular sugar groups from these DTGs, in a way so as not 00:24:13.07 to expose the toxic backbone, which is toxic to the plant also, but also not remove all 00:24:18.00 of them, which produces other toxic compounds. 00:24:21.07 So, this is a story that is ongoing, we're going... we're still working on it, but there's 00:24:25.22 this wonderful digestive duet that's occurring as the caterpillar is removing certain sugar 00:24:31.03 molecules and putting them back on, and putting other molecules back on to protect it and 00:24:36.02 detoxify this molecule as it goes through -- an example of direct defenses. 00:24:41.03 Now, I want to switch to indirect defenses. 00:24:45.05 Now, indirect defenses are based on a concept that probably every politician knows. 00:24:51.06 Now, here's the basic scenario. 00:24:53.22 Here's the plant. 00:24:54.22 And the plan is attacked by Manduca sexta, which is its enemy, right? 00:24:59.02 Now, Manduca sexta is, in turn, attacked by other predators that are all depicted here, 00:25:04.22 there are six of them right here, and they of course are the predators of the herbivore. 00:25:11.11 Now, anyone knows that the enemy of your enemy is your friend. 00:25:17.02 And that is the basis of how indirect defenses work. 00:25:22.09 Indirect defenses, in contrast to the direct defenses, are signals or traits that the plant 00:25:29.24 produces that help predators or parasitoids find and feed on the herbivores that are feeding 00:25:37.02 on them. 00:25:38.23 And that's what that indirect defense looks... how that works. 00:25:41.23 Now, the way it works in Nicotiana attenuata is that, when Manduca sexta begins to feed 00:25:47.06 on an attenuata plant, the plant recognizes it from those FACs that are in the caterpillar's 00:25:52.07 spit and it activates a series of transcription factors, and it activates the production of 00:25:57.05 a beautiful, volatile bouquet, like a Chanel No. 5 that's released not just from the attack 00:26:02.14 leaf but the entire plant. 00:26:04.15 And it's basically just producing this signal that includes a number of molecules, the most 00:26:10.00 important of which is a sesquiterpene called trans-alpha-bergamotene, and trans-alpha-bergamotene 00:26:15.22 attracts this little predator that's down here called Geocoris pallens, a little predator 00:26:20.13 that lives around in the soil on the plant, and it's basically listening, smelling in 00:26:25.06 the air, and when it senses that molecule it knows there's a caterpillar feeding on 00:26:29.17 a plant somewhere. 00:26:30.23 But that little Geocoris also needs local information. 00:26:34.18 Once it arrives on a plant, the plant is big, the caterpillar could be anywhere in the plant, 00:26:39.07 and it utilizes other compounds like these green leafy volatiles on the top there, and 00:26:43.12 particular... particularly the change in a double bond in those green leafy volatiles 00:26:47.23 that gives it local information and allows the Geocoris to be able to localize where 00:26:52.22 on the plant that particular caterpillar is feeding. 00:26:55.07 And, when it gets there to the caterpillar, it just plunges its beak inside the caterpillar 00:27:00.17 and sucks it out and it does that many times. 00:27:04.03 And so what this process is just like calling the police. 00:27:08.12 It doesn't have to do anything more than simply provide accurate, honest information about 00:27:14.20 where a caterpillar is feeding on it, how it's being attacked, and then the predators 00:27:19.05 take it from there. 00:27:21.10 It's a wonderful evolutionarily stable way of dealing with defense because the evolutionary... 00:27:26.19 coevolutionary loop between plant and herbivore is broken by this predator link. 00:27:33.04 Now, we discovered that thanks to the brilliance, really, of the graduate student in the group, 00:27:38.23 Andre Kessler, who is now professor at Cornell, and he invented a predation assay that allowed 00:27:44.04 us to monitor the behavior of this predator in the field under natural conditions. 00:27:48.13 And the predation assay was beautifully simple. 00:27:51.00 He simply just glued eggs of this Manduca onto the bottom of leaves and used those eggs 00:27:59.03 as a monitor for whether or not the predator had come up to the plant. 00:28:02.24 The predator is a very skittish predator. 00:28:05.16 It's called the big eyed bug... it has big eyes, it pays attention to a lot of things, 00:28:10.03 you can't walk up and see, it runs away... and so you need an indirect way to know whether 00:28:14.06 or not it's been around. 00:28:15.21 And yet, when the predator feeds, you can see that it sucks out the egg and it leaves 00:28:19.23 the egg in a nice state behind, and by gluing eggs onto the plant you can see how many predators 00:28:27.06 have come up and visited the plant. 00:28:29.01 And that predation assay had allowed us to be able to work out the transcription factors 00:28:32.17 that regulate volatile production, which volatiles are important, the long- and short-distance 00:28:36.12 signals, all the details of this particular process. 00:28:39.14 Now, it turns out that these indirect defenses don't work alone; they work in synergy with 00:28:46.09 the direct defenses. 00:28:48.04 So, when the cater... when the caterpillar attacks a plant and it causes the plant to 00:28:52.16 produce this wonderful volatile bouquet that is functioning as an alarm call, bringing 00:28:57.24 in predators from long distances away, that will then attack the caterpillars, there are 00:29:03.05 other things going on too, namely that the plant is also producing compounds that are 00:29:08.24 interfering with the digestive process. 00:29:11.06 And these are the protease inhibitors that Jorge Zavala worked on, and the protease inhibitors... 00:29:16.00 here's a seven-domain protease inhibitor... and what they do is they inter... interact 00:29:20.16 with the digestive enzymes of the caterpillar's gut and keeps the caterpillar from digesting, 00:29:25.06 which means that the caterpillar can eat and eat and eat but it doesn't grow, because it's 00:29:28.15 not getting the nutrients. 00:29:30.03 Now, when a caterpillar goes through the stages from being small to large, it becomes pretty 00:29:36.01 immune to this predator, because it's a bratwurst-sized caterpillar at the end and it pretty much 00:29:41.00 can thumb its nose at this little predator who is trying to attack it. 00:29:44.21 But if the plant keeps the caterpillar in a nice, small, vulnerable stage longer, the 00:29:49.20 indirect defense of the predator works much better. 00:29:52.14 So, it's the synergy between direct and indirect defenses that really helps to bruise the... 00:29:58.02 lower the population of caterpillars. 00:30:01.05 Now, there's another type of synergy that occurs as well, and this is depicted very 00:30:05.08 nicely in some videos by Mary Schuman, who is pretending to be a Geocoris predator, sticking 00:30:10.22 a little blue pin up the butt of the caterpillar. 00:30:13.02 And you can see, on a caterpillar that's feeding on a wild-type plant, a wild-type plant that's 00:30:17.11 full of defenses, it's behaving pretty sluggishly -- it's not moving at all when she's poking 00:30:23.17 it, she picks it up with the forceps, it doesn't do any wagging, it just hangs there limp like 00:30:28.01 a doornail. 00:30:29.02 Now, remember this caterpillar is spending a lot of metabolic energy detoxifying the 00:30:34.13 defenses that are in the leaves, the direct defenses. 00:30:39.01 And it doesn't have a whole lot of energy to fight back when it's attacked by predators. 00:30:45.00 Compare that when Mary tries to poke a caterpillar that's feeding on a protease-inhibitor-free 00:30:50.21 plant -- it's got plenty of energy. 00:30:52.11 It's banging around, it's thrashing, and it's defending itself quite well. 00:30:56.24 And that's another example of the synergy between direct and indirect defenses, is that 00:31:04.00 caterpillars that are feeding on toxic plants are lethargic. 00:31:06.15 They are having to spend a lot of energy detoxifying all those metabolites that are going through 00:31:11.22 them, and that slows them down and makes them much more vulnerable to their predators. 00:31:18.00 We so frequently forget because we eat defenseless plants in our normal food supply, we made 00:31:24.09 them defenseless through our agricultural practices, that we forget that eating native 00:31:28.11 plants that are full of chemicals is actually hard, metabolically demanding work. 00:31:34.24 Now, there's another type of direct defense... indirect defense that I want to tell you about. 00:31:39.08 And that's an indirect defense that occurs in the trichomes, which are these little hairs 00:31:43.03 on the surface of the leaves, and you can see as a little droplet appearing here from 00:31:47.11 this magnification of a trichome on the surface of an attenuata leaf. 00:31:50.22 Now, in the trichome is... is a particular type of compound called an acylsugar. 00:31:55.18 Now, acylsugars were thought to be direct defenses, toxins, and there's a good bit of 00:32:01.00 evidence that they are sticky substances that catch insects and sort of tie them up. 00:32:06.13 But... and this was actually first worked on by Alexander Weinhold in the group, and 00:32:11.23 Alexander characterized the structures of these things, and that these acylsugars basically 00:32:17.02 consists of a sucrose molecule and then on each of the hydroxyl groups of the sucrose 00:32:21.10 molecule is esterified a small, short-chain fatty acid. 00:32:26.03 Here are the characteristics of these short... short-chain fatty acids, and these short-chain 00:32:31.06 fatty acids have the smell of baby barf. they're sort of an unpleasant smell and that's the 00:32:36.15 reason why Alexander actually started the project in the beginning, because he had to 00:32:40.08 take care of the caterpillar colony, and he always thought that the caterpillars smelled 00:32:44.15 fairly bad, and... and noticed that when they were feeding on these leaves they were of 00:32:51.03 course eating acylsugars. 00:32:53.02 And when we took these plants to the field... took plants to the field and noticed what 00:32:57.03 caterpillars did when they first hatch out of their egg, we noticed that these... these 00:33:01.05 acylsugars are not defensive at all, they're in fact the first meal of a caterpillar. 00:33:05.11 A caterpillar hatches out of its egg and it begins to lick these... these tops like they're 00:33:10.07 little lollipops, and they get their first meal, and, in the process of getting that 00:33:15.00 first meal, they end up getting a body odor. 00:33:19.00 And the body odor comes from eating those acylsugars and having those fatty acid groups 00:33:24.16 deesterify and come off the body. 00:33:27.09 And so the caterpillar begins to smell of those baby barf fatty acids that are esterified 00:33:33.12 to those sugars. 00:33:34.13 Now, we were very interested to know whether or not smelling attracted the attention of 00:33:39.11 predators that were on the plants. 00:33:41.04 And so we looked at all the predators that occur on plants and none of them cared about 00:33:45.03 this... these baby barf smells -- they didn't seem to respond more to caterpillars that 00:33:48.23 were scented or non-scented. 00:33:50.02 So, we investigated some more. 00:33:52.08 But it turns out that there was another thing that these compounds did to a caterpillar's 00:33:58.21 body odor. 00:33:59.24 Not only did it change the body odor, but it also changed the smell of its poop. 00:34:04.24 There was a caterpillar just pooping there. 00:34:07.03 And poop, when it happens, when it falls, usually falls according to the laws of gravity. 00:34:16.03 It falls down. 00:34:17.03 It doesn't always hit the fan as... as the metaphor goes. 00:34:20.20 And the caterpillar, when it poops, produces a smelly, fresh, redolent poop that falls 00:34:27.14 directly on the ground, and this is Utah where the ground is hot, it's frequently 50 degrees, 00:34:32.16 and those are short-chain fatty acids, so they immediately volatilize, and after five 00:34:36.20 minutes or so they become scent-less and they no longer have that smell. 00:34:41.01 But for five minutes, when the fresh poop has fallen on the ground, it's providing beautiful 00:34:46.01 information to a whole other group of predators. 00:34:49.23 And those are the predators that are walking along in the ground, the lizards and the ants, 00:34:54.19 and it turns out that the lizards and the ants use that volatile information to know 00:34:59.07 that, oops, there's a caterpillar above them, they can just climb up the plant. 00:35:03.03 And you can take fresh frass and dried frass, or you can just isolate the... that... that... 00:35:09.07 those fatty acids and make your own little perfume, which will be available in duty-free 00:35:13.19 shops soon, and call it the scent of the caterpillar, and you can spray it on the ground and spray 00:35:17.16 it on sticks in front of ant nests, and the ants will just come charging up after you've 00:35:21.23 sprayed them, looking for caterpillars. 00:35:24.06 And so, in the end, these trichomes may well be the first meal for the caterpillar, and 00:35:32.05 they're delicious, sugary lollipops, but in the process of scenting their bodies and scenting 00:35:38.10 their frass, they actually turn out to be evil lollipops because they tag them for predation. 00:35:43.16 And that's just another example of how a plant is utilizing indirect defenses to protect 00:35:49.22 themselves. 00:35:51.00 They have very clever ways of bringing in predators. 00:35:54.07 And that was the fourth layer. 00:35:55.23 Now, I'm going to go to the fifth layer, now, and the fifth layer activated by those fatty 00:36:02.06 acid amino acid conjugates that are in the caterpillar's spit. 00:36:05.07 In the fifth layer is a layer of tolerance responses that the plant activates. 00:36:10.15 We had talked earlier in Part 1 about how a plant is a growth machine, fixing carbon 00:36:15.23 dioxide, taking that carbon dioxide, making a whole bunch of metabolites for growth, reproduction, 00:36:21.03 storage, and defense, but at the same time it's also possible to use it to make the plant 00:36:28.10 more tolerant of herbivore attack. 00:36:31.07 Now, until this work, the whole tolerance thing was pretty much a trait-less concept, 00:36:36.01 something that you could look at in populations of plants, but not something you could really 00:36:40.02 nail down to a particular trait. 00:36:42.05 And here we've been able to nail it down to a particular trait. 00:36:45.06 And it came, again, from a field observation. 00:36:47.09 The field observation was that caterpillar-attacked plants, after they plants had senesced and 00:36:53.04 dried out, and then there was another rain, they frequently reflowered -- they produced 00:36:58.00 new flowers after a rain -- but the plants that were not attacked by caterpillars didn't 00:37:02.15 do this reflowering. 00:37:03.16 So, that was an interesting observation. 00:37:05.12 And you sort of wondered, where did these caterpillar-attacked plants get the resources 00:37:09.14 to reflower? 00:37:10.20 This is an annual plant; it should have shut down life, made all the flowers that it could've, 00:37:14.22 and senesced and called it quits. 00:37:16.07 But that's not what they were doing. 00:37:17.21 And I think the answer comes in the life history of the caterpillars that feed on them. 00:37:23.20 Caterpillars go through two stages. 00:37:25.08 They have the eating machine stage, which is depicted right here, where Manduca... 00:37:29.24 Manduca sexta is simply just a larvae trying to consume as much plant material as possible, 00:37:33.18 but then it pupates and molts into this beautiful moth, and it becomes a sex machine. 00:37:39.21 And, as a sex machine, it's no longer eating the caterpillar... eating the plant anymore. 00:37:44.09 And that means that the caterpillar is out of its... out of the concerns of the plant, 00:37:49.13 and the plant, if it had waited and stored resources somewhere else, it was able to reflower 00:37:55.05 and start that whole process over again without having the tissues being lost. 00:37:58.24 And this is what is happening. 00:38:00.19 When... and this is work done by Jens Schwachtje, and his PhD project, and he discovered that 00:38:06.24 the FACs in caterpillar's spit, they elicit a bunkering of photoassimilates into the roots. 00:38:13.20 Now, a plant is a growth machine, right? 00:38:15.22 It's assimilating carbon dioxide from the air and, normally, it would be fixing those... 00:38:20.24 that carbon dioxide into sucrose and sending it from source leaves up to sink leaves to 00:38:25.17 grow more leaf area to make more of a growth machine -- that's what plants normally do. 00:38:30.00 But if they're making more of a growth machine, they're also making more leaves for the caterpillar 00:38:34.03 to eat, grrr... 00:38:35.03 So, you need to stop that process. 00:38:37.12 And when you have a caterpillar on the plant, or you put FACs on a plant, and it doesn't 00:38:42.18 matter where on the plant, the plant, instead of taking that fixed CO2 and sending it up 00:38:48.07 to young sink leaves, it bunkers it down below ground. 00:38:51.15 And it... and Jens was able to show this with some beautiful experiments in collaboration 00:38:56.04 with the Phytosphere Julich, which has a synchrotron, is able to make C-11 carbon dioxide. 00:39:01.09 C-11 has a half-life of 15 minutes, so you have to be right close to the synchrotron 00:39:05.14 -- you can't ship it very far -- and it allows you to look at very short-term partitioning 00:39:10.21 of carbon in a plant after it's fixed and where is it moving and where it moves it around. 00:39:15.14 And here's just some of the data from Jens' work. 00:39:17.23 He was able to show that... up is transport of C-11-labeled CO2 into young leaves, and 00:39:26.03 you can see that it goes... when you just wound and water a plant... and you treat the 00:39:31.14 wounds with water... the fixed carbon dioxide goes up the plant, but if you add spit to 00:39:36.18 the wound it goes down. 00:39:39.08 And it's the specific FACs in that spit that cause it to go down. 00:39:45.13 And he was also able to show that there's a particular subunit of a SnRK kinase which 00:39:49.24 is regulating that. 00:39:51.01 This is this GAL83 subunit that is down-regulated by the FACs. 00:39:55.06 And that's sort of the... the master sink-source regulator, the genetic element that... that 00:40:01.21 is causing this response. 00:40:04.15 And that bunkering, having put that carbon down below ground into the roots, allows the 00:40:09.13 plant to reflower, make bigger flowers, after the caterpillar has gone. 00:40:14.21 So, in many ways this level, this response, this number five, is a man... is the kind 00:40:21.12 of response that Mahatma Gandhi would have against a predator. 00:40:25.17 You just sort of lay low and let it go by, and don't engage in a fight, but just regrow 00:40:32.18 and be able to start again. 00:40:36.17 Okay. 00:40:37.24 The sixth layer and the last layer is probably the most intriguing layer. 00:40:42.19 It's a type of avoidance of this herbivore and it's an avoidance response that has... 00:40:50.11 has to deal with a fairly common natural history problem that... that all organisms have. 00:40:55.16 And that is that some of their interactions are with good guys and some of them with bad 00:40:59.00 guys, and sometimes the good guy and the bad guy are a part of the same genome. 00:41:03.00 So, this moth is a good guy -- it's a pollinator for the plant -- but it lays eggs that are 00:41:09.05 bad guys, that grow into little herbivores that sometimes turn into very big herbivores, 00:41:13.12 that are very disastrous for the plant. 00:41:16.00 And the sixth response has to do with... with dealing with this herbivore by dealing with 00:41:22.07 its mother, its pollinator. 00:41:24.21 Now, I told you in session 1 that this is a plant that attracts that pollinator by producing 00:41:32.23 a compound called benzylacetone, which is depicted up there above the flower, and... 00:41:37.16 and what Danny Kessler discovered is that when the moth is attracted by that particular 00:41:44.22 structure of benzylacetone, not only is it attracted because of the nectar, it nectars 00:41:50.09 and then it oviposits. 00:41:51.17 So, nectaring and ovaposition are linked processes; the more they get nectared by and more visited 00:41:58.16 by this pollinator, the more eggs show up on the plant. 00:42:02.08 The eggs of course turn into herbivores and therefore the more pollination services you 00:42:06.20 get, you might end up getting more herbivores, if the other types of defenses I've talked 00:42:11.12 about earlier aren't effective in cleaning out those herbivores and getting rid of them. 00:42:16.13 Now, we were able to silence benzylacetone production and when we do that we know that, 00:42:21.23 if the plant is not producing benzylacetone, it's pretty much ignored in terms of pollinator 00:42:27.19 activity, and also ovaposition activity by the moth. 00:42:32.05 And Danny Kessler, who is a remarkable photographer but also a remarkable observer of natural 00:42:39.06 history, noticed that attacked plants, when you looked at this... let's do it again at 00:42:43.18 this day night transition... that the plants were beginning to produce a different type 00:42:49.18 of flower after they were attacked. 00:42:52.07 They were producing their normal night flowers, but then they started, when they were attacked, 00:42:55.18 producing a different type of flower that was really only opening in the morning. 00:42:59.24 Now, here is the difference between the morning-open flower on the bottom and the night-open flower 00:43:05.12 at the top. 00:43:06.21 The normal flower is the night-open flower, the one here. 00:43:10.18 And you can see that it opens up in the first night open, and it opens and scents and attracts 00:43:15.22 the moth, and then it closes a little bit for the day, and then opens again and attracts 00:43:19.24 the moth again for the second night. 00:43:22.04 The morning-open flower stays closed that first night. 00:43:25.22 It doesn't scent. 00:43:27.04 And it doesn't attract any moths. 00:43:29.04 And then it opens up just slightly in the next morning, and it attracts a different 00:43:35.13 pollinator, and this is the pollinator -- a hummingbird. 00:43:39.10 And the hummingbird has a very nice characteristic that it lays hummingbird eggs, not caterpillar 00:43:46.08 eggs. 00:43:47.12 And by switching its sexual system to a different pollinator, asking for a different type of 00:43:53.20 postman to bring gametes to you, the plant has basically solved its herbivore problem. 00:44:00.21 And that's pretty remarkable. 00:44:03.07 So, what I've done is told you about all of these changes that occur in the plant when 00:44:10.10 it perceives these compounds here that are in the spit of the plant... the spit of the 00:44:15.13 caterpillar as it chews on... on the plant, and elicits this very complex defense avoidance 00:44:20.14 and tolerance responses. 00:44:22.10 And what I've also told you, I hope... there's basically three messages in behind this remarkable 00:44:28.22 transition that occurs in the plants when it sees these spit factors. 00:44:33.04 The first, of course, is that direct defense is not the only way of coping with herbivores, 00:44:37.10 and most of our agricultural practices dealing with protecting our crop lands have to do 00:44:42.01 with direct defenses -- insecticides that directly kill the crop press... the crop pests. 00:44:48.10 Now, as we've learned from this story, there's many other ways of dealing with your herbivore. 00:44:53.14 And we should be thinking about how to incorporate some of those many other ways into our cropping 00:44:58.11 systems, because some of them may well be much more evolutionary stable than just using 00:45:02.22 direct defenses alone. 00:45:05.10 The second main take-home message that I want to get from this is this interplay of the 00:45:09.08 importance of knowing mechanism so that you can use mechanisms to be able to manipulate 00:45:14.20 function. 00:45:16.12 And when you can manipulate function you can begin to ask, in an unbiased way, what is 00:45:21.03 actually happening in nature between plants and insects, and all the other interactors. 00:45:26.23 And the third main message I want you to get from this is that you can observe an awful 00:45:32.02 lot by just watching. 00:45:33.15 Now, this little tautology is something from Yogi Berra, but I think it applies so cogently 00:45:40.14 to biology today, because we don't teach our students how to watch, particularly not natural 00:45:49.06 interactions, anymore. 00:45:50.06 This is not part of our biological training programs. 00:45:53.08 And so much of the innovation that I've just shown you comes from simple natural history 00:46:00.11 observations. 00:46:01.23 Okay. 00:46:03.11 So, in the third part... that was the end of the second part, in the third part I'm 00:46:08.02 going to talk about seeds, sex, and microbes. 00:46:11.08 In Part 1, I told you that this is a plant that chases fires, it produces seeds that 00:46:16.13 have to live in the seed bank for hundreds of years before the next fire comes along, 00:46:21.09 and I'm going to be talking about how it uses sex to get the best genetic material, to be 00:46:25.24 able to survive that long-time period of... as it waits for the next germination event, 00:46:33.08 and also how it recruits microbes when it does decide to... to... to germinate in opportunistic 00:46:39.08 mutualisms to help protect it against all sorts of stresses that you could hardly predict 00:46:43.14 if you had been in the seed bank for hundreds of years. 00:46:46.07 So, I want to thank you for your attention, but I particularly want to thank both the 00:46:51.04 funding organizations that make this work possible, the long-term, patient funding of 00:46:56.00 the Max Planck Society, and the grants we received from these wonderful agencies that 00:47:01.02 are so unbureaucratic in their administration, and really promote curiosity-driven science 00:47:06.12 in the best way possible. 00:47:07.23 I want to thank the folks at Brigham Young University, particularly Dr. Larry StClair, 00:47:12.22 Ken Packard, and Heriberto Madrigal, that make this wonderful interaction with that 00:47:17.23 remarkable University work, and allow us to use their Lytle Ranch Preserve as a laboratory 00:47:24.09 to study, for the site of these field interactions. 00:47:27.01 And I want to talk... 00:47:28.01 I want to thank all the people who have provided the stunning pictures and movies, the talented 00:47:34.13 photographers and... and folks in the group who have helped support and make some of these 00:47:39.07 slides. 00:47:40.07 And, particularly, Erna Buffie and Volker Arzt, who are really masters of translating 00:47:47.22 science, making beautiful movies, and allowed us to use many of their outtakes from their 00:47:53.03 movies in this presentation. 00:47:55.08 And, you, for your attention.