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.