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Nitrogen Fixing Bacteria: Symbiosis between Bacteria and Plants for Protein Nutrition

Part 1: Symbiosis between nitrogen fixing bacteria and legumes

00:00:02.08 Hi, I'm Sharon Long,
00:00:04.01 from Stanford University.
00:00:05.16 I'm here today to tell you about some remarkable organisms,
00:00:09.09 two kinds of organisms in fact, bacteria and plants.
00:00:13.01 Together, they can do something that neither can do alone:
00:00:16.19 They can bring nitrogen, in its molecular form,
00:00:19.21 which is usually very inert,
00:00:21.24 they can bring that nitrogen into a chemically combined form
00:00:25.07 that can be used for protein nutrition.
00:00:28.11 So, I'll have three topics overall.
00:00:30.24 First, I'd like to give you an introduction to the nitrogen-fixing symbiosis,
00:00:36.14 in particular, between Rhizobium and legumes.
00:00:40.04 I'd then like to focus, in my second segment,
00:00:42.11 on the bacterial genes that are used for the response to the plant,
00:00:46.26 and for the bacteria to be able to provoke the plant,
00:00:49.20 in order to make the home for the bacteria, once they invade.
00:00:55.07 And in the final segment, I'd like to tell you some of our new research
00:00:58.02 on how the plant responds to the signals
00:01:01.15 that come from the bacteria.
00:01:03.21 So now, in the first segment, as I mentioned,
00:01:06.00 we have an overview. I'm going to tell you about
00:01:08.13 something called "root nodules" and how these
00:01:10.24 root nodules form and how they work.
00:01:15.13 To get us started, I'd like to address a subject
00:01:17.24 near and dear to all our hearts, and that is food.
00:01:20.28 When we think about food, we're aware of the need for balance.
00:01:23.16 We need carbohydrates, fats, proteins.
00:01:27.06 And proteins, it turns out, are a particular issue in nutrition,
00:01:32.24 because proteins, unlike carbohydrates and fats
00:01:35.20 for the most part, require a great deal of nitrogen,
00:01:38.26 and nitrogen is scarce.
00:01:41.14 That means that protein malnutrition, the inability to get enough protein,
00:01:46.27 can happen whenever agriculture is deficient or when yields are low,
00:01:52.04 and it's one of the most devastating problems
00:01:54.09 for human health worldwide. So, in Western cultures,
00:01:59.26 when we think about getting protein, we may think,
00:02:02.24 well, I need to have a protein-rich food.
00:02:05.07 Something, which we're going to remember, has a lot of nitrogen.
00:02:09.01 We're going to think about, for example, some poultry.
00:02:11.23 And here they are, poultry, a very protein-rich food.
00:02:16.19 Now, we're thinking about the theme of nitrogen, it's a critical element.
00:02:21.21 Where did these animals get their nitrogen
00:02:23.21 so that they can make protein? They get it from plants.
00:02:26.13 And ultimately, of course, whether we eat meat or eat plants,
00:02:31.16 all human protein ultimately comes from plants.
00:02:36.10 So that raises the question, where do the plants get their nitrogen?
00:02:39.21 The answer is, they get from the soil.
00:02:41.29 Soils include compounds such as nitrates;
00:02:44.28 they will include organic nitrogen, which might be provided
00:02:48.15 from manure or decomposition of plant matter.
00:02:52.19 And the plants will take up this nitrogen,
00:02:54.24 they will synthesize amino acids from the soil nitrogen,
00:02:58.15 and from those, they make proteins,
00:03:01.02 and then we in turn can get our protein from those plants.
00:03:05.07 However, if a field is used over and over again,
00:03:08.24 ultimately it becomes depleted for nitrogen.
00:03:10.25 And that means that this infertile soil is not able
00:03:14.09 to support plant yield or plant nitrogen.
00:03:19.11 Now how do we overcome that?
00:03:21.05 At large scales of agriculture, when fields are used quite often,
00:03:26.18 then the soil the soil can be restored by applications
00:03:30.03 of nitrogen fertilizer, it's usually in the form of ammonium.
00:03:33.29 Now, ammonium, remarkably, can actually be made from
00:03:37.19 molecular nitrogen, which is in the air, which is very abundant.
00:03:41.02 That you may know: The vast majority of the atmosphere is made of nitrogen.
00:03:45.15 But it's incredibly inert.
00:03:47.00 It won't combine with anything, this triple-bond nitrogen.
00:03:51.11 So, in order to coax that nitrogen into a combined form of ammonium,
00:03:57.24 there's a process called the Haber-Bosch process,
00:04:00.26 which is able to take molecule nitrogen,
00:04:04.01 combine it with hydrogen gas at very high temperature,
00:04:08.16 very high pressure, with a metal catalyst, and convert it to ammonium.
00:04:12.21 That ammonium can then be applied on a field.
00:04:16.07 The plants will take up the ammonium, they will make protein,
00:04:18.25 and the yield is therefore supported.
00:04:23.15 But the problem is this:
00:04:25.16 How do we get high temperature and pressure? It's by using fossil fuels.
00:04:31.09 It's mostly natural gas, but other fossil fuels can be used,
00:04:35.20 and we're all aware of the scarcity and the price of fossil fuels,
00:04:40.20 and what lies in the future for their availability.
00:04:44.08 That means that we can't necessarily depend on
00:04:48.04 unlimited supplies of this kind of ammonium fertilizer.
00:04:54.11 Are there alternatives? And it turns out, for millennia,
00:04:58.07 farmers in every continent of the world have known of another way.
00:05:03.09 That is the use of legumes, a very special group of plants.
00:05:07.27 It's a plant family, the Leguminosae, also called Fabaceae,
00:05:12.01 the third largest family in the angiosperms, or flowering plants.
00:05:16.23 Legumes have this remarkable property that they can associate
00:05:20.15 with bacteria from the soil, they can grow a
00:05:24.15 new organ called a root nodule.
00:05:27.11 The bacteria live inside the root nodule, and once the bacteria are there,
00:05:31.20 the bacteria are converting nitrogen into ammonia through an enzyme.
00:05:38.29 This is still energy-intensive.
00:05:41.11 As biochemists, we wouldn't see that it uses highly reduced electrons
00:05:46.09 and a lot of ATP, but is that coming from?
00:05:49.22 It's coming from sunlight.
00:05:51.16 So, the advantage of this is that we're able to nourish plants,
00:05:57.25 and those plants then in turn can nourish the soil,
00:06:01.18 in a process that uses a biological catalyst at room temperature,
00:06:05.12 at ordinary pressure, and uses sunlight to drive it.
00:06:11.28 Now, as I mentioned, farmers throughout the ancient world
00:06:16.13 knew about this. They didn't know why it happened,
00:06:19.24 but they could see that the use of legumes in agriculture to rotate,
00:06:26.25 or to use as a green manure, would assure the fertility of their soil.
00:06:31.15 In the New World, farmers used the common bean,
00:06:35.00 which is a New World plant. In the Mediterranean, they used lupins.
00:06:39.22 In China, they used soybean. And in South Asia and Africa, the cow pea.
00:06:45.18 So again, throughout the world,
00:06:47.22 crop rotation has been used as a key element of sustainable agriculture.
00:06:53.16 So from that view of real nodules,
00:06:56.00 I'd like to draw back just a moment for a cartoon,
00:06:58.29 so that I can describe this in a more simplified way.
00:07:03.10 We know that we're talking about legumes and the way
00:07:07.04 in which they form a symbiosis with nitrogen-fixing bacteria.
00:07:10.15 And one of the key elements of this
00:07:12.23 is that it's able to be used to replace nitrogen fertilizer.
00:07:16.24 How does this happen? So, in a schematic form,
00:07:22.00 we can imagine that there are these bacteria, the Rhizobium bacteria,
00:07:25.16 that grow in the soil.
00:07:26.22 They're able to grow slowly as saprophytes,
00:07:29.03 just scavenging nutrients from the soil.
00:07:32.25 And here's a plant, which, again, could grow if there's fertilizer.
00:07:36.19 But in the absence, it's going to have a hard time.
00:07:40.19 So, if we've got the right bacteria and plant,
00:07:43.21 and I'm going to talk to about that specificity again in a few minutes,
00:07:47.01 they interact to form these:
00:07:48.28 What's shown here in red are root nodules,
00:07:52.07 specialized organs that grow on the root,
00:07:54.14 within which the bacteria are able to fix nitrogen.
00:08:01.00 So, this is where it's all happening. I love this!
00:08:06.17 These are nodules, they're growing on a soybean plant,
00:08:09.02 so you can see some of the nodules here,
00:08:11.07 the growths that are occurring on the root.
00:08:14.05 The plant is up there, and the nodules,
00:08:18.03 once they fix nitrogen and make amino acids,
00:08:21.29 the plant is able to take those amino acids, put it up in the shoot,
00:08:25.17 where it can make chlorophyll, where it can make proteins,
00:08:29.08 and ultimately, to make high-protein seeds.
00:08:34.06 Now I'd like to make several points about this.
00:08:37.22 First, the bacteria in these nodules are fixing nitrogen,
00:08:42.29 but even that is energy intensive.
00:08:45.20 And as I mentioned, ultimately, it's sunlight, through photosynthesis,
00:08:50.17 that allows the plant to support this energy-intensive process
00:08:53.21 of nitrogen fixation. It's occurring only in the root nodules,
00:08:57.28 it doesn't occur anywhere else in the plant.
00:09:00.26 This association is highly specific,
00:09:03.20 and I'll show you that in a moment with a table.
00:09:06.18 It's also established through a complex developmental process,
00:09:09.27 and I'll show you some microscopic views of that.
00:09:14.10 And remarkably, this process is restricted,
00:09:20.15 with one known exception only, in the world.
00:09:23.11 It's restricted to one family, the legume family.
00:09:27.06 We know a little bit about why that occurs,
00:09:30.26 but there's a lot left to find out.
00:09:34.09 The legumes I've mentioned are part of the large family,
00:09:39.05 which includes both agriculturally important
00:09:41.22 and environmentally important species.
00:09:44.24 When we think of high-protein plants, many of the plants
00:09:48.16 that come to mind are in fact the legumes.
00:09:54.12 Both in terms of their seed content and in terms of the content of their leaves.
00:09:59.16 We're familiar with the soybean, cow pea,
00:10:03.02 common bean, all being high-protein seeds.
00:10:06.23 The pea, again, a high-protein seed.
00:10:09.04 Also, alfalfa, which is used as a forage for animals,
00:10:13.17 it has very high-protein leaves.
00:10:15.20 That's why it's so nourishing for animals such as cattle and horses.
00:10:20.08 But beyond the uses for human nutrition, these plants
00:10:26.02 are very important in many natural ecosystems throughout the world.
00:10:30.08 We might think, for example, of this acacia
00:10:33.15 growing up in the African plain.
00:10:36.11 Or we could think of this mesquite tree,
00:10:39.13 let me show you this one over here.
00:10:42.16 And here, a broom plant, a shrub.
00:10:45.27 These legume shrubs and trees often grow
00:10:49.01 in relatively harsh environments, where they provide nutrition
00:10:52.06 for the soil that's around them.
00:10:55.26 So, coming back to the development process,
00:11:00.11 what we want to know is how is it that those incredibly diverse
00:11:05.02 groups of plants are able to establish the association
00:11:11.03 with Rhizobium, and why don't other plants do it?
00:11:15.10 So, I'd now like to tell you a little bit about the specificity.
00:11:21.17 Beyond the specificity for the legume family,
00:11:25.00 within that very diverse family, specific legume types,
00:11:29.22 such as the soybean, are nodulated not just by any old bacterium,
00:11:34.27 but only by certain restricted groups.
00:11:37.12 So here we're looking at the soybean,
00:11:39.08 and here I'm showing you two different species that are known
00:11:42.18 to form symbiotic root nodules on soybean.
00:11:45.12 Here we have the pea. It's a legume also,
00:11:48.11 but its nodulating bacterium is a different one:
00:11:51.04 Rhizobium leguminosarum.
00:11:53.00 I'll have occasion to mention pea and Rhizobium leguminosarum
00:11:56.24 in the second half of my introduction.
00:11:59.20 Here we have a lotus plant called trefoil,
00:12:03.10 and it's nodulated by a different genus, Mesorhizobium, species loti.
00:12:08.12 And the system that I study,
00:12:09.27 which is the genus Medicago, which includes alfalfa,
00:12:13.13 and which is nodulated by Sinorhizobium meliloti.
00:12:17.22 This is the species you'll hear about,
00:12:19.18 in terms of the direct experiments that I describe,
00:12:22.00 through the rest of my talk, with the one comparison
00:12:24.21 that I've mentioned earlier.
00:12:27.06 Now, I've told you that nodule development is complex,
00:12:32.13 but I haven't really shown you what I mean.
00:12:34.09 And so that's what we want to do next.
00:12:36.17 In looking at this, the primary approach that I'll tell you is through microscopy,
00:12:43.21 but embedded in that microscopy,
00:12:46.00 I'll also tell you a little bit about the physiology.
00:12:50.11 So, one of the first changes that occurs when Rhizobium meet a plant,
00:12:56.06 is that the root hairs, which are ordinarily straight,
00:12:59.04 and here's an example of a fairly normal-looking root hair right here...
00:13:03.13 instead, root hairs grow in a way that's deformed.
00:13:07.10 Now don't think of this as root hairs curling.
00:13:10.17 Plant cells don't curve.
00:13:12.09 Think of a plant cell as a balloon inflated really hard inside a box.
00:13:17.18 Plant cells don't curl, but they can grow so that they form a curl,
00:13:21.25 and that's what happens here.
00:13:23.14 If you take a look at this root hair, instead of growing straight,
00:13:26.14 it's grown so that it tops over and makes
00:13:29.17 what's called a "shepherd's crook" or "crozier."
00:13:32.23 In that, you can see a bacterial invasion,
00:13:35.11 which is called an infection thread, and that's shown here
00:13:37.19 at this highly refractile line going down through the root hair.
00:13:44.13 Now, at the same time that invasion is occurring on the outside,
00:13:49.00 early events are also occurring inside the plant root,
00:13:53.09 so I want you to imagine that the plant root is going out this way.
00:13:56.27 Now here we have an uninfected plant root,
00:13:59.01 it's typical plant root cells,
00:14:01.06 you can see they're very long.
00:14:03.29 They look "empty," that's because they're mostly filled with vacuoles,
00:14:08.00 a large vacuole.
00:14:10.15 Plant root cells, in the cortex here, have one main job:
00:14:16.18 That is to transport and transport and transport...
00:14:19.20 water, ions, nutrients, and so forth.
00:14:22.14 That's what these cells do; they don't divide.
00:14:25.18 But if the correct Rhizobium, and this is an example of something
00:14:29.04 that happens only if you have the right species of bacterium and plant...
00:14:32.19 if the correct Rhizobium is infected onto that plant, you get this.
00:14:37.28 You can see the mitotic figures happening here,
00:14:40.22 the cells in the inner cortex of the root have begun to divide.
00:14:45.12 They're going to form what's called a "primordium."
00:14:50.08 Those cells continue dividing,
00:14:52.25 and the infection that started with an infection thread,
00:14:56.13 continues all the way through into these newly dividing cells.
00:15:00.19 Here you can see a cell that's just starting to divide.
00:15:04.00 And what happens is that, if you can follow this arrow,
00:15:08.16 you can see right here, there is a little, membrane-bound,
00:15:13.25 kind of an empty-looking vacuole,
00:15:15.12 and within it, a peach-colored oblong.
00:15:18.17 That is a single Rhizobium bacterium that's
00:15:21.06 being taken up into the host cell, and it's being engulfed
00:15:24.26 by plant plasma membrane that's going to differentiate
00:15:28.28 and provide a special compartment that the Rhizobium
00:15:33.11 will be in when it starts to fix nitrogen.
00:15:37.05 So, the bacteria at this point are released from the infection thread,
00:15:41.05 and they're bound in plant membrane sacs.
00:15:45.08 But that doesn't do justice to what's going on.
00:15:48.27 This bacterium is part of a more elaborate compartment,
00:15:52.00 and I'm showing that compartment here.
00:15:55.26 This, shown in orange, is the bacterium.
00:16:00.28 But you see there's an extra boundary.
00:16:03.06 There's not just the bacterial membrane, but there's another membrane.
00:16:06.24 That's referred to as the symbiosome membrane.
00:16:09.14 It's derived from the plant plasma membrane
00:16:11.29 that was used to engulf the bacterium when it first entered the host cell.
00:16:16.08 But at this point, it's become a distinctive compartment,
00:16:20.04 with its own protein trafficking signals.
00:16:25.00 I'll just tell you a little bit about the basics of transport.
00:16:29.15 Of course, this is the real action:
00:16:32.01 We have nitrogen converted enzymatically into ammonium.
00:16:38.01 This uses ATP and highly reduced electrons,
00:16:41.18 and that ammonium is in turn transported out to the plant.
00:16:46.17 As I've mentioned before, the bacterium gets energy from the plant.
00:16:50.11 Malate, and other dicarboxylic acids, are transported into the bacterium.
00:16:55.29 From there, they enter into the bacteria's own metabolism.
00:16:59.20 So, plant photosynthate providing energy,
00:17:02.29 bacterial nitrogen fixation providing ammonium.
00:17:06.21 That had been suspected for a long time.
00:17:10.02 But emerging data from a number of labs shows that there's more going on.
00:17:14.19 In fact, genetic data show a shuttle involving
00:17:18.09 amino acids that are transported in and out,
00:17:21.22 is also critical to sustain nitrogen fixation.
00:17:25.24 How this occurs is a subject of a great deal of ongoing research.
00:17:31.21 We've got all of these cells, which are packed with bacteria,
00:17:36.08 which are fixing nitrogen.
00:17:37.24 So inside these cells, we've got ammonia created from molecular nitrogen.
00:17:45.11 And over here, you can see the vasculature of the plant,
00:17:49.22 so you can imagine that the stem of the root is going to go up like this,
00:17:55.03 and the stele, the vascular tissue in the root,
00:17:58.26 is going to transport all of those amino acids up to nourish
00:18:02.11 the rest of the plant.
00:18:05.06 So, I've now shown you in this first part of the overview
00:18:09.24 that nodulation occurs by a very complex developmental process.
00:18:15.05 It's species-specific, and the events that I showed you
00:18:18.12 occur only if the correct Rhizobium and correct plant
00:18:21.21 come into combination. Now, in the next segment,
00:18:24.26 what I'd like to do is talk about how the bacteria and the plant
00:18:30.11 create signals that stimulate each other to
00:18:33.05 begin their developmental process.
00:18:35.26 And so in that next slide, we're going to ask this question:
00:18:39.26 How do plants and bacteria use chemical signals for recognition?

Part 2: Function and regulation of Sinorhizobium nodulation genes

00:00:01.02 Hi, I'm Sharon Long,
00:00:02.09 from Stanford University,
00:00:03.19 and I'm here to give my lecture on nitrogen-fixing symbiosis.
00:00:07.03 Today, I'll be talking about the regulation of the genes in the
00:00:10.07 bacterium itself and how those genes are controlled.
00:00:13.23 So just as a review, nitrogen fixation happens in root nodules.
00:00:17.25 These nodules form as a result of action of
00:00:20.12 soil bacteria interacting with a specific plant.
00:00:23.04 Here, you see nodules growing on the root system of a soybean.
00:00:27.15 This is a complicated series of tissue and cell interactions,
00:00:32.02 a complex developmental process,
00:00:33.28 and there's specificity between the bacteria and the plant.
00:00:37.28 I'll talk about one side of that specificity a little later today.
00:00:42.05 In the early stages for sure, and I'll make the case that it's
00:00:45.01 in the later stages as well, bacteria and plant exchange
00:00:49.22 signals in order to coordinate with each other.
00:00:54.10 Now, let's take the standpoint of the bacterium.
00:00:57.01 It's making this huge transition from the free-living bacteria,
00:01:01.01 through invasion, in which the bacteria are able to form
00:01:05.02 an infection thread and penetrate through individual cells,
00:01:08.02 and then into multiple layers of the root, and finally,
00:01:12.08 nitrogen fixation, where the bacteria have become endosymbionts,
00:01:16.17 inside the cytoplasm of a plant cell,
00:01:18.28 and they persist there in a differentiated form, almost like an organelle.
00:01:23.01 All of these changes are accompanied by carefully regulated
00:01:26.24 changes in gene expression.
00:01:31.20 Here's an outline of what I'd like to tell you today.
00:01:34.07 I'll begin by talking about a few tools that we use to study
00:01:37.22 the genetics and the gene expression of the bacteria.
00:01:41.01 And then I'd like to say something about the control of the nodulation genes.
00:01:46.10 This is probably the most studied of any of the gene regulation
00:01:49.19 events in symbiosis. I'll review some of the background,
00:01:53.10 and then I'll talk primarily about the protein activator NodD.
00:01:58.00 I'll then tell you some of our recent work on RNA polymerase
00:02:02.01 and especially the sigma factors and how they may be
00:02:04.19 playing a role in changing and guiding gene expression during symbiosis.
00:02:10.06 I'll finish with several glimpses of how particular sets of
00:02:14.00 genes are controlled, and a preview of all of the factors
00:02:19.24 yet to find in terms of how many genes are active in nodulation.
00:02:27.07 So, the first tool to remind you about is my favorite, and that's genetics.
00:02:33.18 We can take bacteria and, through mutagenesis and then
00:02:37.10 blind screens, we can ask what are the most
00:02:40.18 important genes for symbiosis.
00:02:42.29 Now, there's a couple of different symbiotic phenotypes
00:02:45.08 that we have observed. In contrast to a wild-type situation,
00:02:49.15 where the bacteria growing in the free-living state are able to
00:02:52.17 interact with the correct host plant and make functional nodules,
00:02:56.11 shown here by having a red color reflecting the
00:02:59.17 leghemoglobin that's inside the plant.
00:03:02.09 The nodules harbor bacteria that fix nitrogen, the plants are able to
00:03:06.14 use that to make chlorophyll and protein, they flourish,
00:03:09.10 and they're nice and green.
00:03:10.22 But, if the bacteria have a mutation in a gene that's
00:03:14.14 required for the formation of the nodule, the result
00:03:17.20 will be that the plant forms no nodules.
00:03:19.27 Therefore, it's not getting any fixed nitrogen, and it starves,
00:03:23.12 it's yellow, and it doesn't grow very well.
00:03:26.19 We would call this a nod gene in the bacterium
00:03:30.08 because mutations in that gene result in the so-called
00:03:34.08 Nod- (no nodule) phenotype.
00:03:37.15 The other outcome, again comparing to the wild type,
00:03:40.11 would be that a bacterium might be able to form a nodule,
00:03:43.28 but those nodules don't function, and what I've shown here
00:03:47.05 is I've symbolized that by these empty, white circles.
00:03:52.17 Those show nodules which are not fixing nitrogen,
00:03:55.06 they don't have leghemoglobin, and so we would call
00:03:58.19 this Fix- phenotype,
00:04:01.00 and the corresponding bacterial gene is going to be a fix gene.
00:04:04.07 Another approach is to look at the genome.
00:04:07.15 Bacteria tend to have genomes between about one million
00:04:11.01 for the very smallest pathogens, all the way up to 10 million bases.
00:04:15.20 Rhizobium is on the large size, over 6.5 million bases,
00:04:20.13 divided into three replication units: a chromosome, 3.65 million;
00:04:25.17 and two very large so-called megaplasmids.
00:04:28.11 They're called megaplasmids because they're more than
00:04:30.07 one million bases each.
00:04:32.11 pSymB, 1.68 million, and pSymA, 1.35 million.
00:04:37.18 The nodulation and nitrogen-fixation genes are located on pSymA,
00:04:42.07 but in addition, there are symbiosis genes on pSymB and
00:04:46.01 on the chromosome. So to some extent,
00:04:48.04 the symbiosis genes are dispersed.
00:04:51.10 Now, we can take a genomic approach to look at transcription,
00:04:54.28 and one way in which we have approached that
00:04:57.00 is to do global assays of transcription
00:05:01.06 using a custom-designed Affymetrix chip.
00:05:04.00 On this chip, we place the genome of the bacterium
00:05:07.07 together with 10,000-some genes from the plant
00:05:12.01 as follows: The entire Sinorhizobium meliloti genome is there,
00:05:16.26 every one of the 6000+ open reading frames and the intergenic spaces,
00:05:23.00 and 10,000 tentative consensus sequences from cDNAs
00:05:27.11 that were sequenced from the plant.
00:05:29.13 Now you'll see some experiments later in which
00:05:31.24 we're looking at the expression of genes in Sinorhizobium meliloti,
00:05:36.13 and in one case, we're going to be simultaneously looking
00:05:39.20 at both the bacterium and the plant.
00:05:41.25 And we can do that from a single RNA prep, as shown here.
00:05:46.16 So, if you grind up a nodule,
00:05:49.12 so you're starting with a nodule over here...
00:05:52.06 of course, this is going to be full of many plant cells,
00:05:55.02 each one of those plant cells has bacteria,
00:05:58.00 and those bacteria shown here have differentiated.
00:06:01.15 So a nodule lysate is going to have a
00:06:05.22 mixture of all of those materials.
00:06:09.03 We have a method which allows us to capture both bacteria and plant
00:06:13.29 DNAs at the same time, and that can be shown here.
00:06:16.27 For reference, in the right-hand lane,
00:06:18.29 we have isolated RNA from a plant root,
00:06:22.08 that shows you the large and small ribosomal RNAs here, for reference.
00:06:29.08 On the left-hand lane, we have bacteria grown in culture.
00:06:33.08 You can see again, the bacterial RNAs here.
00:06:36.02 Our prep for nodule RNAs captures both of those, as you can see.
00:06:41.08 And this, by being labeled and hybridized onto our Affy Chip
00:06:44.22 allows us to get a snapshot of both partners at the same time,
00:06:49.28 which is very useful, especially when you're analyzing mutants,
00:06:53.16 and useful because perfect synchrony of nodulation
00:06:58.06 is hard to achieve experimentally.
00:07:01.27 So, having introduced those tools,
00:07:03.25 now I'd like to tell you about the control of the nodulation genes.
00:07:07.21 Just a little bit of review:
00:07:10.08 Nodulation genes here are the genes
00:07:13.24 that encode enzymes that synthesize Nod Factor.
00:07:17.07 I talked about Nod Factor, and I'll show you a cartoon in a moment.
00:07:22.20 It is the substance created by the bacteria that's a
00:07:26.25 molecular signal that causes the plant to form the nodule,
00:07:30.26 and it also causes transcription in a number of other events in the plant.
00:07:35.15 So, powerful signal molecule.
00:07:37.20 The genes in the bacterium that are responsible for producing
00:07:42.20 Nod Factor include the common nod genes, nodABC.
00:07:47.20 The transcription of nodABC is controlled by a promoter
00:07:51.21 that includes what's shown here in this oblong, the so-called nod box,
00:07:56.23 a highly conserved motif that's in the DNA
00:08:00.28 upstream of the nodulation genes, both this set of common nod genes,
00:08:06.05 and others that are coordinately regulated.
00:08:08.26 All of these genes, as I mentioned before,
00:08:11.09 are located on pSymA.
00:08:15.05 Now, this cartoon introduces a couple of themes.
00:08:18.27 First, it reminds us, from my first lecture,
00:08:21.15 that the early events of nodulation include a conversation
00:08:25.24 between the plant and the bacteria.
00:08:28.11 The plant sends a signal out to the bacteria,
00:08:30.28 the bacteria send a signal back.
00:08:33.01 In the case shown on the left, we're taking a look at alfalfa,
00:08:37.01 Meticago sativa.
00:08:39.09 This plant produces a molecule, coded in yellow here,
00:08:43.05 and you'll see the structure in a moment.
00:08:45.01 That signal, from the plant to the bacterium,
00:08:48.03 is able to trigger gene expression, and we'll talk about that in detail.
00:08:52.18 The result of that gene expression, as I mentioned before,
00:08:55.19 is the production of a Nod Factor called NF-Sm.
00:09:00.01 That's the Nod Factor from Sinorhizobium meliloti.
00:09:03.10 It has a backbone of N-acetylglucosamines, which is modified,
00:09:09.02 in the case of Sinorhizobium meliloti,
00:09:11.24 with a 16-carbon N-acyl substitution here,
00:09:15.02 an acetyl, and a sulfate, each shown by a color code.
00:09:19.18 Now, on the right, we have the contrasting example of a cousin,
00:09:24.01 a related legume, pea, or Pisum sativum.
00:09:27.16 Here, we've got the root of the pea plant.
00:09:29.20 It's creating a flavonoid signal whose structure is slightly different,
00:09:33.05 and I've shown that just by the color coding.
00:09:35.04 That signal triggers Rhizobium leguminosarum,
00:09:40.18 which is the symbiont for pea, to transcribe its nodulation genes,
00:09:45.03 and those encode enzymes that make a Nod Factor,
00:09:47.29 but it's a slightly different Nod Factor.
00:09:50.02 The primary difference, which I'm showing here,
00:09:52.04 which is not the only difference, but for the sake of simplicity...
00:09:55.11 A primary difference is the nature of the N-acyl substitution,
00:09:59.07 it's eight carbons instead of 16, and it has four unsaturations.
00:10:03.17 So what we see is some specificity.
00:10:06.07 A signal that goes out from the plant from alfalfa here,
00:10:12.00 or a slightly different one from pea,
00:10:14.07 and the corresponding bacteria which respond to this signal
00:10:17.17 make a slightly different Nod Factor, as shown here.
00:10:20.27 So, this conversation, plant to bacteria and back again,
00:10:24.27 already has some of the hallmarks of the host specificity.
00:10:28.15 We're going to talk only about the first part of that conversation.
00:10:33.09 Here I'm showing again the host plant producing a flavonoid signal,
00:10:38.11 and that is causing transcription in the bacteria.
00:10:41.13 Now, I'd like to focus in on the interaction of the flavonoid
00:10:46.05 with a protein encoded by this gene, NodD.
00:10:50.05 NodD is an activator protein, a part of the LysR family.
00:10:56.17 Now, what are all the elements we're going to have to look at?
00:11:01.17 Of course, at a promoter, there's going to be an RNA polymerase,
00:11:05.20 there's going to be a DNA motif to interact with,
00:11:09.09 in this case there's an activator, and we wonder,
00:11:12.07 how do these plant signals interact with all of these
00:11:15.17 components in order to achieve transcription?
00:11:18.28 And how does this occur in a specific way?
00:11:22.14 So, I'll take a look now first, and most specifically,
00:11:27.10 at the protein NodD.
00:11:31.04 I'll put that here in the context of all of those other components.
00:11:35.00 The red here is showing DNA, all right, so here's the DNA,
00:11:39.23 and the object of this regulation is going to be to
00:11:44.11 achieve transcription of the nodulation genes,
00:11:47.11 so that they can then make the proteins that synthesize Nod Factor.
00:11:52.24 At the DNA, we have a promoter,
00:11:56.01 and that's upstream of the nod boxes.
00:11:58.12 We know that NodD binds to the promoter and bends the DNA.
00:12:03.08 I've shown here a model of NodD as a tetramer;
00:12:07.03 we don't know exactly what its form is,
00:12:09.23 but we suspect that it's most likely to be a tetramer,
00:12:13.04 and more work in the future, we hope,
00:12:15.23 will elucidate that more precisely.
00:12:18.08 We've shown through genetics that the ability of NodD
00:12:21.21 to cause gene expression in bacteria in response
00:12:24.26 to a plant signal requires the chaperonins GroES and GroEL.
00:12:29.19 Also, we found through genetics that this entire system
00:12:32.09 requires the "stringent response," that being a response,
00:12:36.19 classically to starvation, where a protein, here RelA,
00:12:44.13 causes the synthesis of guanosine tetra- or pentaphosphate,
00:12:48.09 shown here as this star.
00:12:52.28 All of that has to interact with RNA polymerase,
00:12:56.20 the machine that's going to interact with DNA
00:12:58.27 and cause template-driven transcription of an mRNA
00:13:02.25 off of the DNA template.
00:13:05.12 So, these are some of the things that we know,
00:13:08.05 but there's a lot of unknowns.
00:13:11.00 Let's ask, for example, how the inducer works with the NodD.
00:13:18.18 Now I'm not going to show you the data on our GroEL work,
00:13:22.10 but I will just mention that we have a combination of
00:13:25.18 genetic data and in vitro biochemical studies
00:13:29.23 that show how GroEL is required for NodD and luteolin to be active.
00:13:35.18 In one of the experiments that follows,
00:13:37.04 I'll be referring to that some more.
00:13:39.08 Let's now just ask about flavonoids,
00:13:42.17 including the particular flavonoid luteolin, and NodD.
00:13:48.11 So in this experiment, we're doing the following:
00:13:51.28 We're growing Sinorhizobium either without an inducer
00:13:55.23 or with an inducer, so it either has just culture medium
00:13:59.10 or it has the plant signal in it.
00:14:01.25 From those cultured cells, we purify NodD,
00:14:05.08 and we ask, using electrophoretic mobility shift assay,
00:14:09.04 does that NodD bind to the nod box promoter or not?
00:14:14.05 So in pairs here, you're going to be seeing,
00:14:17.03 in the open boxes, how much DNA binding is accomplished
00:14:23.07 by this amount of NodD onto the nod box promoter
00:14:28.20 (the nodulation genes' upstream region).
00:14:31.16 And this is the amount of binding when the NodD in question
00:14:35.11 came from cells grown without inducer.
00:14:37.25 But you can see here that, if the NodD had been purified from
00:14:43.03 cells grown with inducer, the same amount of NodD
00:14:46.27 does much more DNA binding.
00:14:50.02 So it appears that the affinity of NodD for its DNA target
00:14:54.26 is increased if the NodD was purified from a
00:14:58.11 cell exposed to the plant inducer luteolin.
00:15:03.26 Now, this is an in vitro study.
00:15:06.15 In this case, all of the NodD is being purified from
00:15:09.13 cells grown with no inducer.
00:15:12.18 That purified NodD is then incubated with buffer only
00:15:19.18 or with the plant inducer luteolin.
00:15:22.27 So we're going to have "minus" for no luteolin and
00:15:25.16 "plus" for added flavonoid luteolin.
00:15:29.27 We found early on in data not shown here,
00:15:32.28 that if this were done with NodD only, there was no result.
00:15:38.17 However, if we carried this experiment out in a system where the
00:15:42.13 NodD was also presented with GroEL/GroES,
00:15:47.24 magnesium, ATP, and inducer, there was a profound effect.
00:15:52.26 And that's shown here.
00:15:55.16 The primary data are as follows:
00:15:58.09 In each case, we've got purified NodD that was incubated
00:16:02.06 just with magnesium, ATP, and GroES/GroEL here,
00:16:07.06 or with those same components and luteolin;
00:16:11.14 just chaperonins and ATP here, or with luteolin;
00:16:17.10 components only, or with luteolin, and so forth.
00:16:19.23 And what you can see is that the presence of the luteolin,
00:16:24.05 the flavonoid inducer, is able to enhance the ability of purified NodD
00:16:29.24 to bind to its target DNA sequence in the nodulation promoter.
00:16:36.28 So, what we've got here, then, is a demonstration that,
00:16:42.18 in the presence of chaperonins
00:16:44.26 and in the presence of the flavonoid inducer,
00:16:48.22 again we see enhanced ability of NodD to bind to DNA.
00:16:52.13 And that's quantified here, if you look at the fraction bound:
00:16:55.17 absence of luteolin in the incubation mixture,
00:16:58.14 presence of luteolin.
00:17:00.24 Now, this was exciting to us because we were starting
00:17:04.11 to get at the question of how the flavonoids
00:17:07.26 affect the bacteria's ability to express genes.
00:17:11.01 But recall, I told you that one of the hallmarks of this system
00:17:14.19 is that it is specific.
00:17:16.07 We know that different plants make different flavonoids.
00:17:18.26 Some of them work in vivo, and some don't.
00:17:22.02 Is that due to DNA?
00:17:24.06 Knowing that we should expect any in vitro regulatory system
00:17:30.13 to explain specificity as well as other aspects of NodD activity,
00:17:36.05 we carried out an in vivo experiment based on some earlier data
00:17:39.26 from some years ago, that showed a correlation
00:17:44.20 between what species a NodD protein came from,
00:17:50.14 and what kinds of flavonoids a cell would respond to.
00:17:54.05 So in this experiment, genetically,
00:17:56.25 we've got a responder cell that's going to report for us
00:18:00.23 whether nodulation genes are transcribed or not.
00:18:03.17 It's got a nod gene promoter fused to beta-galatosidase.
00:18:08.23 But that genetic construct has its own nodD taken away.
00:18:13.06 There's a deletion for nodD, but there's a nod-lac fusion.
00:18:17.05 Into that strain, we're going to place one or the other
00:18:20.20 of these two plasmids.
00:18:22.15 These plasmids have the same vector, so the same backbone.
00:18:26.24 They also have a promoter that's exactly the same;
00:18:30.09 it's a constitutive promoter that's highly expressed.
00:18:34.16 What's different in them is that, in this strain,
00:18:37.17 we've got a gene that represents the trimmed open reading frame
00:18:41.11 for NodD from Sinorhizobium meliloti, which is the symbiont of alfalfa.
00:18:46.24 Now in this other plasmid, we have the trimmed open reading frame
00:18:51.05 of NodD from Rhizobium leguminosarum trifolii,
00:18:53.23 which is a symbiont of clover.
00:18:56.06 So everything is the same except the actual amino acid sequence
00:19:01.19 of the two NodDs.
00:19:03.12 Now we're going to place those into a reporter strain
00:19:06.06 that has no NodD of its own,
00:19:08.17 and ask whether nod genes are expressed or not.
00:19:12.04 So in the left-hand column here, what you're seeing
00:19:15.02 is whether or not nod genes are expressed in response to luteolin.
00:19:19.26 And what you can see is that when the cell includes
00:19:24.17 this plasmid that has the Sinorhizobium meliloti nodD,
00:19:28.28 then yes, there is nod gene expression.
00:19:33.10 However, if the strain has the nodD gene that came from Rhizobium trifolii,
00:19:39.10 then it's kind of iffy, there's not very much expression.
00:19:43.26 So this suggests that this NodD can induce
00:19:48.14 in response to this molecule, but this NodD cannot induce very well.
00:19:54.18 But now let's ask:
00:19:56.01 How do those reporter strains respond to a completely
00:20:00.23 different flavonoid, shown here?
00:20:02.23 You can see for example it has only one hydroxyl,
00:20:05.12 whereas luteolin has four.
00:20:08.12 Well, now, if we take the strain containing the Sinorhizobium meliloti
00:20:11.29 NodD, we see that there's no expression at all.
00:20:15.10 Whereas the NodD from the Rhizobium trifolii shows good activity.
00:20:20.27 So this shows that the amino acid sequence of just NodD
00:20:25.21 is sufficient to account for a different response
00:20:29.12 to one flavonoid structure versus another flavonoid structure.
00:20:33.23 We wanted to put that together with the data that I just showed you,
00:20:37.21 that demonstrated that NodD can bind better to its
00:20:44.12 nod box target if it's got luteolin present.
00:20:48.22 But what we found was something of a surprise.
00:20:51.29 Now what I'm showing here on the top are the
00:20:54.00 structures of the flavonoid that I've introduced before,
00:20:56.19 that's luteolin, and a set of flavonoids here
00:21:00.27 that in vivo do not cause gene expression in Sinorhizobium meliloti.
00:21:07.17 So we would infer from that that the NodD of Sinorhizobium meliloti
00:21:11.26 cannot interact productively with any of these,
00:21:14.20 because they don't work in vivo.
00:21:17.00 However, if we carried out experiments similar to
00:21:20.06 those I showed you before with each of these,
00:21:23.06 we got the following:
00:21:25.04 Here is a set of data, and here's another.
00:21:30.08 This for daidzein, that's the molecule up here.
00:21:33.14 This one for eriodictyol, which is the molecule there.
00:21:36.14 And what you can see here is that,
00:21:38.16 if you compare NodD protein incubated with components such as
00:21:43.07 GroEL, magnesium, ATP,
00:21:45.19 or those same components plus the flavonoid in question,
00:21:49.26 these flavonoids, even though they don't work in vivo,
00:21:52.24 they still enhance the binding of NodD to its target promoter.
00:21:57.02 That's true for daidzein, that's true for eriodictyol.
00:22:00.19 And what this shows is that DNA binding is caused
00:22:04.21 even from flavonoids that are not active.
00:22:10.13 And some of the examples of the data
00:22:13.25 and a quantification are shown here.
00:22:16.06 This shows a gel shift of NodD from Sinorhizobium meliloti
00:22:22.19 presented with buffer only,
00:22:25.29 with daidzein, luteolin, and naringenin, three inducers,
00:22:31.25 of which only the luteolin is really active in vivo.
00:22:35.01 Nonetheless, all of them cause gel shifting,
00:22:38.01 and that's quantified here.
00:22:39.16 What you can see in this graph is NodD with no inducer
00:22:44.24 of any kind added, and NodD with the three different inducers,
00:22:48.20 some of which are active in vivo and some of which aren't.
00:22:51.22 So right now, our working model is that NodD
00:22:56.00 is able to bind DNA with an increased affinity
00:22:59.19 no matter what flavonoid is there.
00:23:02.02 And in fact, the flavonoids that do not work in vivo
00:23:06.24 may actually bind to NodD and inhibit the ability
00:23:10.19 of the correct flavonoid to induce,
00:23:13.07 by causing unproductive DNA binding.
00:23:16.03 So DNA binding does not explain the specificity of NodD
00:23:21.21 for the flavonoid inducers that come from different plants.
00:23:25.15 That leaves us with a series of new experiments
00:23:27.24 to do in the future, and among them,
00:23:30.20 what we want to ask is:
00:23:32.11 Does the presence of the correct flavonoid
00:23:35.03 somehow affect the interaction of NodD with RNA polymerase?
00:23:39.29 And that's led us in turn to ask about the components
00:23:44.21 of RNA polymerase, and in the end,
00:23:47.18 we hope also to work in vitro with the guanosine tetraphosphate
00:23:52.20 and ask about all of these components together.
00:23:56.03 But in this next section, I'll just like to talk about RNA polymerase.
00:24:00.08 In particular, I want to talk about the sigma factors.
00:24:03.23 Now just as a review, RNA polymerase in prokaryotes has
00:24:09.01 a core made of two α subunits (two copies of the α subunit),
00:24:14.04 a β and β' subunit, and in addition, a sigma factor,
00:24:18.20 which has multiple domains, and they're shown here in this cartoon,
00:24:22.28 which is derived from E. coli sigma factor.
00:24:25.18 And what we now know from work in many labs over the years,
00:24:29.13 is that there are many kinds of sigma factors.
00:24:33.10 Originally, the one that was best characterized
00:24:36.01 was the housekeeping, or rpoD, σ70 type of sigma factor.
00:24:41.14 And that's the form of sigma factor that reconstitutes
00:24:45.13 with RNA polymerase for the transcription of most genes
00:24:48.22 in E. coli and in other organisms.
00:24:51.05 The sigma factor here has various alpha-helices
00:24:56.10 and other subdomains; that's what these letters are for.
00:24:59.10 But this is one polypeptide as it interacts with different parts
00:25:02.27 of the β', β, and α subunits.
00:25:06.23 But in addition to the rpoD,
00:25:08.27 there are other sigma factors, a stress sigma factor rpoS,
00:25:12.24 which is very prominent in survival responses of E. coli;
00:25:17.05 heat shock rpoH;
00:25:19.00 in some enterics, there's also rpoN,
00:25:22.07 which is important for nitrogen metabolism and for other functions
00:25:26.11 such as nitrogen fixation in Klebsiella;
00:25:29.11 and a number of other sigma factors,
00:25:32.13 and I'm going to come back in a few minutes to one in particular, the rpoE.
00:25:39.04 In Sinorhizobium meliloti, our annotation showed
00:25:42.19 that there's at least double that number of sigma factors.
00:25:46.09 One of the striking differences with enterics
00:25:48.10 is that there's no copy of rpoS.
00:25:51.21 However, there is a housekeeping sigma,
00:25:54.11 it looks very comparable to the σ70 of E. coli.
00:25:58.25 There are two copies of the rpoH or so-called heat-shock sigma.
00:26:04.11 There are ten copies at least of rpoE,
00:26:07.16 in addition to the fecI originally identified for relationship to iron control,
00:26:13.16 and the rpoN that's important for nitrogen fixation.
00:26:17.09 In the following section, I'd like to tell you some of our
00:26:20.15 new work on rpoH and refer briefly to
00:26:23.23 some emerging work on rpoE.
00:26:28.09 We wanted to know what do those two different copies of rpoH do?
00:26:34.12 Are they important for heat shock?
00:26:36.08 Are they important for symbiosis?
00:26:38.10 Let's start with a look at how these are expressed.
00:26:42.02 On the left-hand column, you can see how the two different copies,
00:26:46.11 rpoH1 and rpoH2, are expressed in free-living cells.
00:26:51.26 The rpoH1 tends to be expressed mostly
00:26:55.22 in late log (late exponential) and stationary,
00:27:00.02 but rpoH2 is expressed mostly in stationary cells.
00:27:04.18 If you grind up nodules and ask whether
00:27:06.20 these rpoH genes are transcribed,
00:27:09.09 you can see that rpoH1 is transcribed, yes, it's present in nodules;
00:27:14.12 but rpoH2 was not detectable in our assays.
00:27:18.10 That leads to surprise in a minute, as I'll tell you.
00:27:22.04 Knocking out one or the other or both of these rpoH genes
00:27:27.09 leads to some phenotypes in the free-living state.
00:27:31.09 The rpoH1 mutant is very sensitive to high temperature.
00:27:36.11 rpoH2 does not appear to have much of a phenotype,
00:27:40.04 but the double mutant, similar to the rpoH1,
00:27:43.07 is affected by high temperature.
00:27:46.15 Here's the surprise about the symbiosis.
00:27:49.25 What we found was that a mutation in rpoH1 was Fix-.
00:27:56.11 rpoH2, if it's knocked out only by itself,
00:28:00.10 does not appear to have any symbiotic defect.
00:28:03.11 However, if you knock out both rpoH1 and rpoH2,
00:28:07.28 and despite the fact that rpoH2 doesn't seem
00:28:11.11 to be detectable as a transcript in nodules,
00:28:13.29 remarkably, that's Nod-.
00:28:16.09 That means that one or the other of those rpoH sigma factors
00:28:21.14 has to be functioning in order for the symbiosis
00:28:24.25 to proceed past some of the very earliest stages.
00:28:28.12 So that's a mystery that we are beginning to look at in more detail.
00:28:33.08 Meanwhile, we've also asked about the spectrum of genes
00:28:36.01 that are expressed.
00:28:38.12 In the following experiment, we used the Affymetrix chip
00:28:41.13 that I described earlier.
00:28:44.17 And what we're going to be doing here is taking a look
00:28:47.16 at the transcripts in wild-type cells, and in each of the two single mutants,
00:28:53.27 and the rpoH1H2 double mutant, as we grow them in culture,
00:28:58.24 give them a heat shock, and then harvest the
00:29:01.07 RNA to assess global transcription.
00:29:05.09 Now, in the following, I'll show you summaries of our data.
00:29:10.01 Remarkably, we found that, when you heat shock wild-type rhizobia,
00:29:15.15 almost a third of the genes in the genome, over 2000 genes,
00:29:19.16 are affected one way or another.
00:29:21.11 One thousand of them go up, another thousand go down.
00:29:25.13 Another intriguing feature of this transcription picture is shown here.
00:29:29.18 Now, if you take the entire genome of Sinorhizobium and you look
00:29:32.27 at the distribution of open reading frames,
00:29:34.23 you can see that more than a half of the open reading frames
00:29:37.11 are in the chromosome, and that's shown in this here,
00:29:41.13 so that's the chromosome.
00:29:43.20 pSymB accounts for this many, as a fraction of the whole,
00:29:48.10 and this is the number that are in pSymA.
00:29:51.14 That's the total genome annotation,
00:29:53.18 but if you look at those 2000 genes that are changed
00:29:56.22 one way or the other by heat shock,
00:29:58.27 what you see is that the chromosome is underrepresented,
00:30:02.15 and the two Sym plasmids are overrepresented.
00:30:05.00 So that, in and of itself, was kind of intriguing,
00:30:07.22 especially when you put it together with the fact that the
00:30:10.03 double mutant of those sigma factors is very defective in symbiosis.
00:30:17.13 Here was another surprise:
00:30:19.19 If you take a look in this picture only at the genes
00:30:23.13 that are increased in heat-shocked cells, and that's, as I said,
00:30:27.23 over a thousand, what we find is that the large majority
00:30:33.15 of those are increased by heat shock
00:30:37.21 even if the two rpoH sigma factors are both knocked out.
00:30:42.22 And that's shown here.
00:30:44.06 These are the increased genes by heat shock in a wild-type,
00:30:48.17 and this blue circle shows those genes
00:30:52.10 that do not get enhanced in a mutant.
00:30:57.20 These are the genes that do not get enhanced
00:30:59.27 by heat shock in an rpoH1H2 double mutant.
00:31:04.01 And the overlap is only about 200.
00:31:08.29 So, only a minority of the heat-shock genes are apparently
00:31:13.16 controlled by the rpoH sigma factors.
00:31:16.23 So, they're doing other things,
00:31:19.09 and some other regulatory system is taking the job that,
00:31:22.19 in E. coli, would be done just by rpoH.
00:31:25.27 Another surprise that needs to be followed up.
00:31:29.24 So I'd now like to ask the question:
00:31:33.14 How about the other sigma factors?
00:31:35.25 As we move forward looking at rpoH,
00:31:37.27 we know that there are more sigma factors to
00:31:40.13 account for in Sinorhizobium meliloti.
00:31:43.20 Now one that's important for sure, that I won't say more about,
00:31:47.24 is obviously the rpoN σ54 sigma factor known to be
00:31:53.04 required for the transcription of nitrogenase and a
00:31:55.29 number of the other genes in the late differentiated state.
00:32:00.10 The rpoE family presents some other very intriguing puzzles.
00:32:06.09 Now, rpoE was identified in E. coli as a sigma factor
00:32:12.21 responsible for genes that were triggered by denaturation
00:32:18.05 of proteins in the periplasm.
00:32:20.14 They're controlled post-transcriptionally by an anti-sigma factor,
00:32:25.04 which captures them in an inactive form,
00:32:27.15 unless the anti-sigma factor is somehow triggered
00:32:30.21 to release the rpoE sigma,
00:32:33.10 which can then go and associate with core polymerase
00:32:36.16 and turn on a large number of genes.
00:32:38.27 So we began to look genetically at these rpoE genes.
00:32:42.27 We found that the single mutants in each of the rpoE
00:32:49.11 open reading frames don't have any particular phenotype,
00:32:52.14 and that's shown here. Right?
00:32:53.27 Single rpoEs have some slight membrane sensitivity, but basically,
00:33:01.29 they seem normal in culture and they're normal for symbiosis.
00:33:06.08 We've now also gone through and made all of the double mutants
00:33:09.15 for each of those sigma factors.
00:33:12.00 What we find there is that some of these rpoE sigma factor
00:33:16.03 double mutants have a symbiotic defect.
00:33:20.01 They make nodules, but nodules do not fix nitrogen.
00:33:23.15 That suggests that somewhere in the transition between the
00:33:27.17 beginning of nodule formation and the differentiation of the bacteria,
00:33:31.25 essential genes require the activity of some of the rpoE sigma factors.
00:33:37.28 It also points out the likelihood that these are at least partially redundant;
00:33:41.29 that's why none of the single mutants had a phenotype,
00:33:44.18 but some of the doubles do.
00:33:47.16 So, in our future work, we are now asking,
00:33:51.29 which of the genes are controlled by each of these rpoEs,
00:33:55.18 and can we use transcriptional analysis of the global picture
00:33:59.14 of rpoE control to understand the redundancy
00:34:02.10 and understand where they're required in symbiosis.
00:34:06.09 So, from that view of the RNA polymerase,
00:34:10.25 I'd like to go to a few more case histories.
00:34:13.18 But we need to keep in mind that whatever stages we're looking at,
00:34:18.14 we may need to consider the global picture of RNA
00:34:22.04 polymerase and of other regulatory contexts
00:34:25.14 to understand how specific regulatory circuits
00:34:28.23 are able to function in symbiosis.
00:34:31.07 Now, my next topic is about the control of
00:34:33.18 extracellular polysaccharides.
00:34:36.01 EPS, extracellular polysaccharides, are required for symbiosis.
00:34:41.04 And I'll tell you in this section about how these are
00:34:43.27 regulated by a family, that includes a periplasmic regulator
00:34:49.25 and a two-component regulatory system.
00:34:52.11 Now, the EPS in Sinorhizobium meliloti has been studied
00:34:55.16 in detail by Graham Walker and his colleagues at MIT,
00:34:58.24 over a number of years.
00:35:00.11 The name of this exopolysaccharide is succinoglycan
00:35:03.20 because it is modified by acidic groups,
00:35:06.01 including succinyl groups.
00:35:08.15 And they have shown that mutants which are not able to
00:35:11.26 synthesize succinoglycan are able to provoke nodules
00:35:17.04 to form on alfalfa as a host, but they cannot invade.
00:35:21.11 So what's shown here, which is a root hair with bacteria invading,
00:35:26.15 and these bacteria are all labeled with green fluorescent protein,
00:35:30.27 allowing you to follow their invasion into the plant.
00:35:34.25 This process will not occur unless you have the
00:35:38.05 extracellular polysaccharides.
00:35:41.04 Now here's some mutants that were found by Graham Walker
00:35:44.27 and his colleagues that were able to make polysaccharide,
00:35:49.19 but they don't make the right amount.
00:35:51.05 In fact, they make too much.
00:35:53.00 So what you're seeing here are bacteria that are streaked
00:35:56.17 on a solid medium including a dye called calcofluor.
00:36:00.06 Calcofluor in solution is not fluorescent,
00:36:02.27 but when it binds to certain beta-glucans, it becomes fluorescent.
00:36:06.28 It's used sometimes as a laundry brightener because,
00:36:10.12 among other glucans, calcofluor can bind to cellulose,
00:36:13.24 which is the constituent of cotton.
00:36:16.11 And when it binds and becomes fluorescent,
00:36:18.28 that means that any cotton fabric that has calcofluor
00:36:21.26 will fluoresce slightly in sunlight, because there's a little bit of UV in sunlight.
00:36:26.17 Picks up the UV, fluoresces bright blue,
00:36:29.10 that makes the laundry look whiter than white.
00:36:32.07 Now in the case of Sinorhizobium,
00:36:33.26 it's lucky for us that the succinoglycan also binds calcofluor.
00:36:37.29 So a wild type, which you can see here, has a light,
00:36:42.03 white glow when it's grown on calcofluor medium
00:36:47.20 and exposed to UV light.
00:36:50.21 But two mutants, here exoR mutant that's defective,
00:36:56.08 and here an exoS mutant that's constitutively active
00:37:00.22 (that is, it's hypermorphic), each of these
00:37:04.09 makes too much polysaccharide.
00:37:06.13 And actually, it turns out that's not good for symbiosis either.
00:37:09.12 If a bacterium makes too much polysaccharide,
00:37:11.23 it doesn't do very well at invasion.
00:37:14.07 So what's going on here?
00:37:15.28 Why does this mutant
00:37:18.06 make too much polysaccharide when it's overexpressed,
00:37:20.24 and this mutant makes too much polysaccharide
00:37:23.08 when it's defective?
00:37:24.16 Well, we would guess that that's because this is a negative regulator
00:37:27.11 and this is a positive regulator.
00:37:29.22 But we took these and found, fortuitously,
00:37:34.03 some unexpected phenotypes that went beyond polysaccharide.
00:37:39.01 Here's the first of those:
00:37:41.15 And that is that mutants defective in exoR
00:37:45.22 or overproductive of/overactive in exoS are not motile.
00:37:51.13 They are not able to swarm on a motility plate, and that's shown here.
00:37:56.16 This is wild type; you can see that on this particular agar,
00:37:59.09 it's able to swim around, it makes a big, wide colony.
00:38:03.19 But the exoR- mutant or the exoS overexpression mutant,
00:38:07.25 both of these are not able to swarm.
00:38:10.12 That's because they're not motile.
00:38:12.12 This plate also lets me introduce to you two genetic constructs
00:38:16.07 that give us clues.
00:38:18.07 In the first one, over here, we've taken this exoS mutant,
00:38:22.04 and we've taken a wild-type ExoR (that's what we
00:38:27.09 infer to be an inhibitory factor)...
00:38:31.01 we've taken ExoR and we're overexpressing it.
00:38:34.04 If we overexpress that ExoR in the presence
00:38:39.08 of this overexpressed ExoS,
00:38:42.12 it apparently allows the cells to be normal again.
00:38:47.01 So that suggests to us that ExoR actually inhibits ExoS,
00:38:53.10 and even if you have a hyperactive ExoS,
00:38:56.00 if you then add more and more ExoR,
00:38:58.18 you're able to get things back to the wild-type balance.
00:39:02.19 Now, the second clue was also very interesting.
00:39:05.23 Here's exoR, and in a population of exoR mutants,
00:39:12.03 which are, as you can see, nonmotile,
00:39:14.02 a suppressor arose.
00:39:16.08 That suppressor allows the exoR to be motile again.
00:39:20.06 So when we tracked down the gene and sequenced it,
00:39:22.27 we found that that suppressor was actually in a gene called chvI.
00:39:29.25 ChvI is a DNA-binding protein that's linked to ExoS,
00:39:34.28 which is a response regulator.
00:39:36.25 ExoS and ChvI are a two-component system.
00:39:40.10 And without even asking about ExoS,
00:39:42.26 what we did was to mutate ExoR and find a suppressor
00:39:47.16 that was in the substrate of ExoS.
00:39:52.24 Now, motility appeared to show us a linkage of the exoS
00:39:58.10 and the exoR with the chvI,
00:40:01.21 but how about other phenotypes?
00:40:04.02 Well, one of the other dramatic changes that happens
00:40:08.19 in an exoR- or exoS constitutive bacterium
00:40:16.02 is that genes which are normally upregulated in a wild type
00:40:22.24 are very low in expression in those mutants.
00:40:26.17 And you can see that here, if you take a look here,
00:40:29.26 these blue panels.
00:40:31.12 These are actually two duplicates of an exoR-
00:40:34.28 and an exoS constitutive, showing a whole set of genes
00:40:39.08 that are not appropriately regulated if exoS
00:40:42.18 and exoR are in those defective conditions.
00:40:47.08 The two suppressors that I showed you,
00:40:49.04 however, the ones that reverse motility?
00:40:52.09 They also reverse the transcription defects
00:40:57.00 and restore them to wild-type levels,
00:40:59.07 and that's shown in these panels on the right,
00:41:03.04 where you can see that coordinate restoration
00:41:08.27 of a wild-type function from the same suppressors.
00:41:13.07 So, what we find is that the motility defect
00:41:18.06 and in the transcription profiles, exoS and exoR and chvI
00:41:24.01 all appear to be targeting coordinated functions.
00:41:29.16 We believe, based on some biochemical data,
00:41:33.00 that the system works as follows:
00:41:36.01 We've shown biochemically that ExoR is in the periplasm
00:41:41.01 in between the cytoplasmic membrane and the outer membrane.
00:41:44.23 So as a negative regulator that's present in the periplasm,
00:41:48.04 it's unique at this point.
00:41:50.19 There's no model for how this might be working,
00:41:53.09 because there's no other proteins of that type yet reported.
00:41:57.07 ExoS and ChvI are the two-component system,
00:42:02.14 with ExoS as a histidine kinase and ChvI is the
00:42:06.27 response regulator that binds to DNA.
00:42:09.25 And through ChvI, all of these functions,
00:42:12.15 including motility, transcription, and we've also shown biofilm formation
00:42:16.05 and other functions... they're all controlled.
00:42:19.23 Now, this presents us with a lot of mysteries.
00:42:23.04 For example, his-kinases (sensor kinases) are presumably
00:42:28.17 responding to some kind of change in the environment,
00:42:31.00 some signal. What is it?
00:42:33.16 Is the plant sending something?
00:42:36.14 Are other bacteria sending something?
00:42:39.01 What are the factors in the
00:42:41.06 environment that control the activity of ExoS?
00:42:45.12 ExoR as well, how does that act?
00:42:47.25 Does that also respond to the environment?
00:42:50.09 So these are some of the mysteries that we hope to
00:42:52.16 work on at the molecular level,
00:42:55.22 and one of the other directions we're going right now
00:42:58.04 is to look very specifically for the targets of the ChvI DNA-binding protein.
00:43:05.05 Now, ExoR and ExoS, as I showed you, control a lot of genes.
00:43:10.21 There are more genes out there.
00:43:12.17 Now we already know that this regulon is required for exopolysaccharide,
00:43:18.18 it has to be just right in order to get invasion,
00:43:21.06 you saw that there are many genes that are controlled.
00:43:24.11 But we also think that, as you go from invasion
00:43:27.05 all the way to differentiation,
00:43:29.08 there are many more genes than that.
00:43:32.17 First, in a promoter trap screen, genetically,
00:43:35.26 we found several dozen bacterial promoters
00:43:39.08 that appear to turn on in between the beginning of invasion
00:43:43.15 and the final differentiation.
00:43:45.10 So, we're looking at those genes to ask,
00:43:47.26 what is it that controls them?
00:43:50.16 Furthermore, we've recently carried out more genetic screens
00:43:54.24 of the type that I described in the beginning --
00:43:57.11 putting random bacterial mutants on plants and asking whether
00:44:00.28 those bacteria are able to cause nodules to form
00:44:04.18 and whether those nodules are functional --
00:44:06.25 to find more nodulation mutants and fixation mutants.
00:44:09.29 We have already found several new genes
00:44:12.25 not previously recovered in earlier screens,
00:44:15.10 for transporters for example,
00:44:17.20 which suggests that the bacteria must be able to import or perhaps
00:44:21.15 export materials in order to accomplish nitrogen fixation
00:44:24.29 and differentiation. And in addition,
00:44:29.14 we've taken the transcriptional approach as follows:
00:44:34.07 So based on our Affymetrix transcription studies,
00:44:37.23 we were able to show that hundreds of bacterial genes change,
00:44:43.01 if you compare cultured bacteria to bacteria in nodules.
00:44:46.08 Over 300 genes increase in expression, including the obvious suspects,
00:44:52.00 such as nitrogenase and fixation genes.
00:44:55.06 But many more than that, 900-some genes, go down.
00:44:59.02 These decreased genes include biosynthetic functions
00:45:02.25 such as amino acid synthesis and others.
00:45:06.02 So the bacteria is having a major change in its gene expression profile.
00:45:11.10 The plant changes as well.
00:45:14.06 Now, several hundred genes change,
00:45:17.27 of which 250 or so increase in the expression,
00:45:21.17 and many plant genes decrease in their expression as well.
00:45:25.20 Now what we wanted to do was ask something about
00:45:28.08 the strategy of how the plants and the bacteria
00:45:31.13 control their transcription in these nodules.
00:45:33.29 So what we're going to do in the next set of experiments
00:45:37.12 is compare the transcripts in a wild-type nodule, such as this one here,
00:45:42.12 to a nodule that's being established on a wild-type plant
00:45:46.13 by a bacterium that's mutated.
00:45:49.08 Now what mutant are we going to use?
00:45:51.00 We're going to use a mutant called fixJ.
00:45:53.27 Now the protein FixJ is required for bacteria in
00:45:58.12 the nodule to transcribe the nitrogenase, or nif genes.
00:46:02.16 And that works as follows:
00:46:04.00 Leghemoglobin, which is present in the plant cytoplasm,
00:46:07.13 shown in this cartoon, buffers oxygen
00:46:10.06 so that the free oxygen tension is very low
00:46:13.01 (although oxygen is delivered at a high rate by leghemoglobin).
00:46:17.06 Now, the low oxygen is able to interact with
00:46:20.12 the two-component regulatory system, FixL and FixJ,
00:46:23.29 that activates a signal transduction cascade
00:46:27.00 that results in nitrogen fixation.
00:46:30.07 So, here's the plant cytoplasm
00:46:32.07 and the bacterium responding to the plant's signal.
00:46:35.14 If you have no FixJ in the bacterium, then there's no nitrogen fixation.
00:46:41.17 So the bacterium provokes a nodule, it gets into the plant,
00:46:45.25 but then it doesn't work.
00:46:47.18 So the RNA transcription studies we're doing now
00:46:51.24 are going to compare a nodule made with wild-type bacteria
00:46:56.07 and a nodule that's made and infected
00:46:58.27 with bacteria that then don't fix nitrogen.
00:47:01.02 And here are results.
00:47:02.28 Let's take a look at the wild type first.
00:47:05.27 As I mentioned... and by the way,
00:47:09.02 we're looking only at the transcripts that are going to go up.
00:47:13.21 The plant has about 250 or so transcripts that are upregulated in a nodule.
00:47:19.22 The bacterium has somewhat over 300 transcripts that increase.
00:47:25.19 This is the wild type.
00:47:27.08 Now, if we ask,
00:47:29.00 what happens if we look at the transcripts
00:47:31.05 made in a nodule that was created by the mutant?
00:47:35.10 We see that a nodule made by the fixJ mutant
00:47:41.01 upregulates very few transcripts.
00:47:44.11 The vast majority of these 300-some transcripts that are
00:47:48.07 increased in bacteria when they're wild-type
00:47:52.05 apparently requires the FixJ system.
00:47:56.26 So I would say from this that the bacterial transcription profile
00:48:01.14 is responding to some signals right in time,
00:48:06.02 at the point that nitrogen fixation is occurring.
00:48:09.22 We found a contrasting result when we ask these same nodules
00:48:14.18 that have so few bacterial transcripts,
00:48:17.03 how many plant transcripts do they have?
00:48:19.25 And that's shown here.
00:48:21.01 What you can see is that almost all of the wild-type transcripts
00:48:25.16 shown in this outer box are also made in the nodule
00:48:29.20 that resulted from the bacteria that is mutated for nitrogen fixation.
00:48:35.27 So I would say that suggests a couple of things.
00:48:40.20 First, that the plant isn't making its decision about
00:48:47.04 whether to transcribe these genes at the last minute.
00:48:50.28 It made that decision to upregulate these genes early on,
00:48:55.03 before the bacterium makes the decision about
00:48:57.19 whether or not to fix nitrogen, right?
00:49:00.07 So most of these genes, I would guess,
00:49:03.08 are being made to build the nodule in the first place,
00:49:06.20 that the strategy of the plant is to decide on the morphogenesis.
00:49:11.08 But it also shows that, yes, there are some genes,
00:49:14.07 and they're shown in this outer part that's not
00:49:16.12 covered by the overlap,
00:49:19.26 there are some genes that do depend on bacterial nitrogen fixation,
00:49:24.02 in order for the plant to express them.
00:49:25.26 So we want to find out in future studies
00:49:27.29 how those are controlled, as well as how some
00:49:32.05 of these are controlled irrespective of the bacterial signaling.
00:49:37.17 And that brings me to the final topic,
00:49:41.15 in which we've been able to use a combination of
00:49:44.20 bacterial genetics and plant genetics,
00:49:47.13 as a way of elucidating new signals.
00:49:50.22 In a screen of plant mutants, we asked,
00:49:54.21 can we find plant mutants that fail to accomplish nitrogen fixation,
00:50:00.27 fail to accomplish correct symbiosis?
00:50:03.20 Just as a baseline, let's take a look here at wild type.
00:50:07.06 Now, in a wild-type plant with wild-type bacteria,
00:50:10.23 you get nice nodules, here's those nodules.
00:50:13.23 If you do a section through a nodule,
00:50:15.10 you can see all of the infected cells.
00:50:19.04 So here in the corner is the vasculature of the plant,
00:50:21.29 so the root is going like this.
00:50:23.23 Here's the nodule coming out and all of these dark-stained cells
00:50:26.17 are packed full of successful bacteria fixing nitrogen.
00:50:31.20 Using a bacterial strain that has a glucuronidase fusion
00:50:35.08 in the nif genes, but is still wild-type,
00:50:38.19 we're able to see that, if you take this kind of section of a nodule
00:50:42.28 and you stain it for glucuronidase activity,
00:50:45.11 you get this deep-blue stain, and that means that the nitrogen fixation
00:50:49.06 genes are being transcribed.
00:50:51.21 But now let's take a look at a plant mutant.
00:50:55.15 So this plant mutant is called dnf,
00:50:57.28 that stands for "defective in nitrogen fixation."
00:51:00.28 If you see here, the bacterial action (wild-type bacteria)
00:51:07.05 on this plant yield tiny, little, white nodules.
00:51:11.10 They're not functioning, they're not fixing nitrogen.
00:51:15.08 A look inside those nodules shows that the bacteria do get into cells.
00:51:20.02 Here's the vasculature again, here's the root,
00:51:22.20 here's the nodule.
00:51:24.05 All these dark-staining cells are packed full of bacteria.
00:51:27.14 However, if you put in the indicator for nif to glucuronidase
00:51:32.17 and you stain for glucuronidase activity,
00:51:35.00 you can see here that the nif genes (nitrogenase genes)
00:51:38.10 are not being expressed. There's no blue stain.
00:51:42.11 So something about this plant mutant
00:51:46.27 is not allowing nitrogen fixation to occur.
00:51:49.20 So we can say that the bacteria need whatever it is that DNF1 does
00:51:55.08 in order to differentiate and fix nitrogen.
00:51:58.21 Having carried out the crosses and mapping necessary,
00:52:04.25 we have cloned and identified DNF1.
00:52:07.24 We found that DNF1 encodes a plant protein
00:52:12.17 that is a nodule-specific signal peptidase.
00:52:16.09 Now, using this illustration from Molecular Biology of the Cell,
00:52:20.16 I can just go through roughly what we would
00:52:23.15 expect the signal peptidase to do doing.
00:52:25.23 In the plant cytoplasm, you're going to have the endoplasmic reticulum.
00:52:30.23 Some plant proteins are going to be encoded in the nucleus,
00:52:34.17 they're going to be translated such that they go
00:52:37.06 into the lumen of the endoplasmic reticulum,
00:52:40.08 and that is made possible by a signal peptide.
00:52:43.15 The signal peptidase cleaves off the signal peptide as so,
00:52:49.01 releasing the mature polypeptide product.
00:52:54.27 Now, what do those do?
00:52:56.28 I'll get to that in a moment.
00:52:58.05 But, what we just conclude is that there's going to be something
00:53:03.11 about the action of DNF1 that's going to result
00:53:09.07 in the production of a mature protein that might go to the cell wall,
00:53:15.00 or it might go into vesicles.
00:53:18.12 So whatever is being produced by the DNF1 signal peptidase
00:53:24.16 may be purposed for secretion or for vesicle delivery.
00:53:32.00 So what are the substrates?
00:53:33.29 And here, our work was able to intersect
00:53:36.14 with the work of Eva Kondorosi and Peter Mergaert
00:53:38.29 and their colleagues.
00:53:40.21 They had discovered a family of nodule-specific plant proteins
00:53:44.10 that they called NCR, "nodule-specific cysteine-rich proteins."
00:53:50.20 Using our mutant, they have been able to show that these NCR proteins
00:53:55.09 are actually processed by our DNF1 signal peptidase.
00:54:00.28 Therefore, what we can envision is that the lumen
00:54:03.12 of the endoplasmic reticulum, as a donor compartment,
00:54:06.25 is going to be full of these NCR peptides after they're processed.
00:54:14.01 And these may be then delivered to a target compartment;
00:54:18.04 we might envision that as having the Rhizobium.
00:54:21.14 So, once again, what can consider here
00:54:24.05 is that we've got NCR proteins from the plant
00:54:28.26 being processed into the endoplasmic reticulum, put into vesicles,
00:54:35.00 and then delivered into a compartment where
00:54:37.18 they actually go into the Rhizobium and
00:54:40.12 cause those Rhizobium to differentiate.
00:54:43.06 This model can be tested in a number of ways,
00:54:46.04 and those are underway at present.
00:54:50.20 What we can envision though, is the following:
00:54:54.04 We know that these bacterial cells,
00:54:57.23 which are here shown in a closeup of an infected cell in a nodule,
00:55:02.14 these bacterial cells must be getting a lot of signals from the plant.
00:55:06.21 So if we envision this as a cartoon of a plant cell,
00:55:10.06 and here's the cytoplasm of the plant cell,
00:55:13.02 we already know from previous work that the leghemoglobin
00:55:16.27 that I mentioned earlier, that gives the nodule its pink color,
00:55:20.12 helps to buffer oxygen.
00:55:22.25 It keeps free oxygen low while delivering it at a fast rate,
00:55:26.15 and we know from earlier work that there's a signal transduction
00:55:29.19 cascade that triggers the activity of NifA and
00:55:35.03 eventual transcription of nitrogenase, due to the FixL/FixJ
00:55:40.00 oxygen-sensitive two-component system.
00:55:44.07 But now what we know is that something else is needed.
00:55:47.29 Unless you've got DNF1 processing the NCRs
00:55:53.01 and maybe doing other jobs as well,
00:55:55.02 unless you've got DNF1, you still won't get nitrogen fixation.
00:56:00.10 So, a completely new kind of process.
00:56:06.13 Deep inside the nodule, there is a new signaling process
00:56:10.12 going that we've now discovered.
00:56:12.26 And this raises the possibility that specific
00:56:16.22 DNF1-processed peptides may be triggering differentiation.
00:56:23.08 As Mergaert and Kondorosi have shown,
00:56:25.14 these peptides are also able to carry out control
00:56:30.23 of bacterial cell division and DNA endoreduplication.
00:56:34.20 So there are many hundreds of NCRs,
00:56:37.14 and tracking down what each of them does
00:56:39.11 is a big challenge for the future.
00:56:41.15 From our standpoint, as we look at bacterial differentiation,
00:56:44.26 we want to know, are these effects direct or indirect?
00:56:49.01 Does an NCR peptide actually go into the bacterium
00:56:53.17 and interact with a receptor, or is it causing some condition
00:56:56.25 in the plant that then causes gene transcription
00:57:00.23 to change in the bacterium?
00:57:02.19 So, our purpose is right now to characterize the mechanism
00:57:06.27 in the plant of how these DNF1 signal peptidase components
00:57:12.26 are working to ask whether there are other substrate
00:57:15.28 proteins as well, and to find out their mechanism in symbiosis.
00:57:20.13 So what I've shown you today is an outline of how
00:57:23.04 we're taking a look at bacterial genes from the early
00:57:26.11 nodulation processes all the way through the differentiation
00:57:29.26 of the bacteria that makes it possible for them to fix nitrogen.
00:57:34.09 And I think in particular that the use of plant mutants,
00:57:40.06 as we did with DNF1, to look for plant factors
00:57:44.11 that control bacteria may be an especially promising
00:57:46.24 direction for the future.
00:57:49.25 They're going to be many levels of regulation,
00:57:52.03 from external signals to sigma factors.
00:57:55.02 I think that the combination of genetics and in vitro transcription
00:57:59.21 studies are important.
00:58:01.26 And a lot of our effort in the future is going to be directed towards
00:58:05.22 this double study of plant mutants and bacterial genes,
00:58:10.11 in order to try to dissect out the signals and controls.
00:58:14.07 So with that, I'll conclude this part of the lecture,
00:58:17.02 and my third lecture is going to be on how the plant responds
00:58:20.24 to the bacterial signals at the various stages of nodulation.
00:58:25.20 Thank you.

Part 3: Plant genes and cell response in nitrogen-fixing symbiosis

00:00:00.00 Hi, I'm Sharon Long,
00:00:01.20 from Stanford University,
00:00:02.18 and I'm here today to talk about our recent on the plant side
00:00:05.20 of the nitrogen-fixing symbiosis, specifically about
00:00:08.20 genes and cell responses.
00:00:11.17 By way of review, I'll mention again that the Rhizobium symbiosis
00:00:15.21 happens in organs called root nodules.
00:00:19.15 And within these nodules, bacteria are able to fix nitrogen
00:00:23.23 and provide that to the plant for nutrition.
00:00:26.05 This is complex developmental process,
00:00:29.02 it goes through a number of stages,
00:00:30.17 and you'll see some of those today.
00:00:32.07 It is species specific, which I've discussed in some of my earlier talks.
00:00:36.11 And importantly, for today's lecture,
00:00:39.20 bacterium and plant each respond to signals from the other.
00:00:44.03 An outline of my talk today is shown here.
00:00:46.08 At the beginning, I'll give you some introduction,
00:00:48.12 reviewing some of the dynamics of how bacteria and plants interact.
00:00:52.12 Then I'd like to talk about some recent work in our lab,
00:00:55.10 first about infection and about of group of proteins called flotillins,
00:01:00.04 and also about a Nod Factor receptor from the plant.
00:01:04.00 Then I'd like to talk about two plant mutants, dnf1 and dnf2,
00:01:08.08 which we've identified as being defective in late stages of nodulation.
00:01:13.19 And by describing the genes that we've now cloned from those mutants,
00:01:17.21 I can share with you some of our ideas on how the symbiosis is working.
00:01:23.11 So, here I'm showing a review of the basic idea
00:01:27.22 of signal exchange in the early phases of nodulation.
00:01:31.22 The plant secretes a flavonoid signal,
00:01:35.03 this flavonoid acts as a trigger in the bacterium,
00:01:38.09 and that causes the transcription of genes called nod genes,
00:01:42.05 which encode enzymes. Those enzymes are able to
00:01:45.05 synthesize this second signal, the so-called Nod Factor, down here,
00:01:50.16 sometimes abbreviated as NF in some of the slides to come.
00:01:53.29 This is a modified chitin fragment,
00:01:57.08 you can see there's four residues of N-acetylglucosamine.
00:02:01.28 These are modified at the reducing end with a sulfate,
00:02:06.04 and at the non-reducing end with both an acetyl and an N-acyl group.
00:02:11.11 And in one of the slides yet to come,
00:02:14.11 I'll be talking about the differences between the plant response
00:02:17.24 to chitin to a true Nod Factor.
00:02:21.05 So Nod Factors are very powerful,
00:02:23.12 and this gives you an idea of just how powerful they are.
00:02:26.18 On the top, we have an alfalfa root, and on this root,
00:02:30.14 Sinorhizobium meliloti, the symbiont, has established a nodule,
00:02:34.28 and this is an intact nodule.
00:02:36.15 It's been cleared, and you can see the deep brown color,
00:02:40.08 which is from the metals that are in the various cytochromes
00:02:43.17 and enzymes of the bacteria while they're fixing nitrogen.
00:02:47.09 Now at the bottom, we see a form that looks very similar,
00:02:51.01 and yet this has no bacteria in it.
00:02:53.09 This entire structure on an alfalfa root
00:02:55.27 was caused just by a small droplet of Nod Factor.
00:03:00.27 So this one chemical is able to produce an entire organ on the plant.
00:03:07.18 Here is another one of the characteristic early events
00:03:10.19 in Rhizobium-legume interactions.
00:03:13.23 This shows root hairs on the root of a plant, and here on the right,
00:03:20.00 you can see a normal root hair, which grows straight.
00:03:22.07 And here you see a root hair that has been provoked
00:03:25.22 by Rhizobium to grow in a such a way that it tops over
00:03:30.11 and forms a curl. And here in the crook is where the
00:03:35.11 bacteria are trapped, and these deformed root hairs
00:03:40.18 of various kinds are very characteristic of the effect
00:03:44.05 of the correct Rhizobium on its compatible host.
00:03:49.04 Now, the bacteria are then able to travel by proliferating
00:03:55.13 and burrowing their way into the plant cell itself.
00:03:59.16 And this white arrow shows an infection thread;
00:04:02.18 this is formed by the plant in response to the bacteria.
00:04:06.10 Within the infection thread, bacteria are proliferating,
00:04:09.21 and they are invading through the root hair cell,
00:04:13.02 and as you'll see, eventually down into further cell layers.
00:04:17.17 So there's the infection thread, and you'll see more about that in a little while.
00:04:22.04 I'd now like to show you something about the dynamics
00:04:24.20 of how Rhizobium affect plants.
00:04:27.08 Let's start by looking at normal root hairs.
00:04:29.19 So, on a root, the youngest part of the root is over here,
00:04:32.29 and you can see that they don't have any root hairs yet,
00:04:35.12 and then the root hairs get longer.
00:04:37.15 And over here, they're full sized.
00:04:40.02 So, root hairs get to a particular size, and then they stop.
00:04:43.23 Now let's watch in this movie,
00:04:45.17 to see how root hairs elongate normally, that's without Rhizobium.
00:04:50.11 So here you see root hairs.
00:04:52.01 They start, they grow, and then at some point
00:04:55.05 they reach a mature length, and they just stop,
00:04:58.24 but they grow fairly straight during this entire process.
00:05:02.27 Now we're going to look at root hairs on a plant that
00:05:05.20 has been treated with its Rhizobium symbiont.
00:05:09.21 So here, the root hairs are in the process of growing,
00:05:12.23 and if you watch what happens to them as they grow,
00:05:16.18 you'll see how different it is from unperturbed growth.
00:05:22.25 So here, for example, we see root hairs that are curling.
00:05:28.23 Over on this side, if you look at this root hair, you can see that it's growing,
00:05:33.00 and then it pauses, see that pause?
00:05:35.17 And then it emerges, and now it pauses and branches again.
00:05:39.18 So there's two things going on.
00:05:41.11 One is this characteristic pause, so right here you can see
00:05:46.01 there's a pause right before it starts to top over.
00:05:49.05 And the other is that you're getting branching or curling,
00:05:53.03 other kinds of deformation.
00:05:55.02 So Rhizobium has a profound effect on the morphogenesis
00:05:58.06 of this particular plant cell.
00:06:01.19 Now, we've seen a little bit now about root hair deformation,
00:06:05.00 and I'll mention along the way that Rhizobium can also
00:06:09.09 cause roots to engage in specific transcription,
00:06:13.00 which we study with microarrays,
00:06:14.22 and they provoke cell division.
00:06:17.00 But for the moment, I just want to look a little bit more
00:06:19.14 about what happens to these root hairs,
00:06:22.01 because if you think about it, root hairs are on the surface of the plant,
00:06:26.10 they're single cells, they have a high surface-to-volume ratio...
00:06:30.00 it makes sense that these are going to be
00:06:32.11 exposed to the signals from the outside,
00:06:34.11 and they might be the place where signal transduction begins.
00:06:38.03 So, we might ask, by cell biology and by genetics,
00:06:43.22 are there receptors, and what is the signal transduction pathway?
00:06:47.23 And ask whether we can study root hairs
00:06:49.29 as a way of capturing those.
00:06:51.19 Now, signal transduction is often a complex and fairly rapid
00:06:56.12 multiprotein process, so we set out to look for events
00:07:00.12 that occur in root hairs that are cell autonomous and fairly rapid.
00:07:06.17 One of the earliest studies that we did in this area was
00:07:10.19 simply to look at the electrochemical potential across the plant membrane.
00:07:15.28 So in this, we're taking a single root hair here,
00:07:20.00 and by putting in a microelectrode and measuring the difference
00:07:23.13 in potential between the interior of the cell and exterior of the cell,
00:07:26.29 we were able to find that untreated root hairs have a stable,
00:07:31.29 very negative potential at about -130 to -140 mV.
00:07:37.08 However, if we treated a root hair with Rhizobium,
00:07:40.21 what we found was that the Rhizobium and its Nod Factor
00:07:45.15 are able to cause a depolarization of that plasma membrane,
00:07:49.29 so that the potential change across the membrane here diminishes.
00:07:55.00 And that happens actually within a minute.
00:07:58.05 In our studies and others', this has been shown to be accompanied
00:08:01.15 by ion fluxes (ion currents) near the tip.
00:08:05.11 Now one ion in particular is very interesting to us,
00:08:08.09 and that is calcium. We've used different techniques,
00:08:11.14 but here's one of them that we have used.
00:08:13.29 On the left, you can see a root hair,
00:08:17.03 and into this root hair we're delivering a mixture of
00:08:19.16 two different fluorescent proteins.
00:08:21.07 One is fluorescent irrespective of the calcium concentration,
00:08:25.01 the other increases its fluorescence when calcium is high.
00:08:28.27 Using those two together, we can take a ratio
00:08:31.20 and get the value of calcium corrected
00:08:34.03 for the concentration of the cytoplasm.
00:08:37.03 And then we can track that over time.
00:08:39.17 So now we're going to follow calcium in this slide.
00:08:42.29 Now, this is a pseudo-color representation
00:08:45.23 of the fluorescence corrected for cytoplasmic concentration.
00:08:50.08 Cool colors mean low calcium, warm colors mean high calcium.
00:08:55.19 If you take a look at the root hairs here, they're all blue,
00:08:59.17 that means low calcium.
00:09:00.28 As you move in time, and these are ten second intervals,
00:09:04.07 you see the warmer color, yellow, appearing from the tip of the root hair,
00:09:09.07 so calcium is getting high at the tip.
00:09:12.27 As you continue the time series up here,
00:09:15.29 you can now get to a point where you see this extremely high calcium,
00:09:19.25 shown by red, and this happens to be in the part of the
00:09:23.02 cell represented by the nucleus.
00:09:26.06 So, what can we say, and I'll quantify that with the following graphs.
00:09:31.02 First, if you compare an untreated cell, that's here,
00:09:34.17 and here's the baseline of the calcium, with the presentation of
00:09:38.07 one nanomolar of Nod Factor,
00:09:40.24 what you find is that about ten minutes after the presentation
00:09:44.01 of the Nod Factor, you get these sharp upswings in calcium.
00:09:49.01 And that's what's going on here.
00:09:51.25 This is the calcium spiking represented there.
00:09:55.13 Now, number two, if you put in a higher amount of Nod Factor,
00:10:00.29 10 nanomolar, now you get something more complex.
00:10:06.01 You get a faster response, and this turns out to be the
00:10:10.21 tip flux of calcium coming in, and then calcium spiking
00:10:15.10 happens at the same time.
00:10:18.00 Now the other data that I'll show you here is that,
00:10:21.21 if you take chitin alone...
00:10:25.04 Now remember I pointed out that Nod Factor is like an
00:10:28.28 oligomer of chitin N-acetylglucosamine resides,
00:10:32.24 with some modifications. But what if you take the modifications off?
00:10:36.01 It's not Nod Factor anymore, it's just chitin.
00:10:38.26 If you add very, very large amounts of chitin,
00:10:46.29 we've got one micromolar here, so a thousand-fold higher,
00:10:50.10 you get some spiking, but you need much more chitin than Nod Factor,
00:10:56.16 and it's not completely normal.
00:10:58.06 So for that, we can say that the plant is
00:11:02.15 exquisitely tuned to the Nod Factor.
00:11:05.24 Finally, we do know that the calcium flux and calcium spiking
00:11:10.13 are not just separated in space, but they're really independent
00:11:17.03 in that you can add high Nod Factor after calcium spiking
00:11:23.16 has started, and you'll get the big influx.
00:11:26.27 So they really do appear to be two separate events.
00:11:31.04 Now a third response that we were able to document
00:11:34.06 very early in nodulation is this one:
00:11:37.02 Using a whole seedling assay and with an indicator
00:11:42.00 that shows the presence of hydrogen peroxide,
00:11:45.00 we can follow normal roots over time,
00:11:49.11 and we can assess how much peroxide are they producing.
00:11:52.26 And in a normal, untreated root hair, that's shown here.
00:11:56.14 They don't produce zero, they produce a modest amount,
00:11:59.08 and here's what it looks like over a period of about 1.5 hours.
00:12:03.00 Now what if we treat those with Nod Factor?
00:12:05.19 What we find, very intriguingly, is that Nod Factor causes
00:12:10.00 the rate of evolution to be diminished.
00:12:13.18 So a lower level of hydrogen peroxide is produced
00:12:18.29 in the presence of Nod Factor.
00:12:22.09 Now what if we add an "elicitor," pathogenic elicitor?
00:12:26.16 We would expect an elicitor to cause the plant to
00:12:29.18 mount a defense response, and sure enough,
00:12:31.23 if you look at the amount of hydrogen peroxide coming in the plant
00:12:35.19 after the treatment with the elicitor, it's accelerated.
00:12:39.13 So it seems that roots are able to elaborate hydrogen peroxide,
00:12:45.24 an example of reactive oxygen species,
00:12:49.05 that Nod Factor diminishes that,
00:12:51.18 which would suggest it's lowering its defenses,
00:12:54.01 and an elicitor increases it, consistent with an increase in defenses.
00:12:59.26 Now, this is only over the couple of hours,
00:13:02.18 we're interested here only in very early events.
00:13:06.05 In fact, the defense reactions appear to have a very interesting
00:13:11.14 and complex role later on in nodulation, but this is not related to those.
00:13:17.14 This is just in the first two hours or so.
00:13:20.25 So now we can fill in a bit what we know
00:13:22.28 about the early responses of the plants to the rhizobial Nod Factor.
00:13:28.16 We know that it causes nodules to form, cell divisions in the plant.
00:13:36.25 We now can fill in that, in addition to the overall root hair curling,
00:13:41.16 which we could see in the microscope,
00:13:44.21 other kinds of assays demonstrate that there's a rapid depolarization
00:13:48.23 across the plant plasma membrane in response to Nod Factor.
00:13:52.23 This is accompanied by calcium flux.
00:13:55.10 Slightly later there's calcium spiking in the cytoplasm and
00:13:58.13 a suppression of the rate of reactive oxygen production.
00:14:03.13 Now one other topic that I'll just mention briefly is transcription.
00:14:08.24 We've been able to assess transcription during nodulation,
00:14:13.13 at various stages, with a specialized approach,
00:14:18.16 which is shown on the next slide.
00:14:21.01 We, following our work on determining the complete genome
00:14:26.14 sequence of the bacterium, we then constructed an Affymetrix chip
00:14:32.05 in which we had the complete bacterial genome,
00:14:34.24 plus about 10,000 sequences representing probable genes
00:14:40.16 from EST libraries in the plant, and we put
00:14:43.17 the two genomes on the same Affymetrix chip.
00:14:47.21 So we call this our SymbioChip, and through analysis of RNA
00:14:54.00 species from the nodules, we can actually get a readout of
00:14:57.18 both bacteria and the plant at the same time.
00:15:00.27 And through that, we were able to show that,
00:15:04.07 within the first 24 hours after treatment with Nod Factor or
00:15:09.26 after treatment with bacteria, a characteristic set of plant genes
00:15:15.01 are upregulated or are downregulated.
00:15:18.10 Some four dozen or so sequences,
00:15:21.23 and I'll talk a little bit more about that in a moment when
00:15:24.02 I get to the mutant analysis.
00:15:26.19 But now, let me put all of those events on a timeline for you.
00:15:30.21 So I'm going to start here on the left.
00:15:32.27 Now, we know that in the very early stages, within a minute,
00:15:36.16 we get calcium flux, suppression of reactive oxygen,
00:15:41.29 we get a depolarization.
00:15:43.21 There's also calcium spiking at about 10 minutes.
00:15:46.17 There are morphological changes.
00:15:48.15 You saw in the film that the root hairs pause
00:15:51.28 and swell a little bit right before they start to curl.
00:15:55.00 Then there's morphogenesis of the root hair,
00:15:57.22 forming branches and curls and both.
00:16:01.13 And we can see the cell divisions. Finally, the transcription.
00:16:05.26 Now here's the timeline for that happening.
00:16:08.14 If we start at time zero, and this is where the bacteria are being added,
00:16:12.25 those first events, such as the calcium flux and the depolarization,
00:16:18.05 happen within a few minutes, one to a few minutes.
00:16:21.02 Calcium spiking at an average of 10 minutes.
00:16:24.10 The gene expression that I mentioned, that we can assess
00:16:26.20 with our Affymetrix chip, that's happening as early as a
00:16:30.14 few hours and goes on for about 24 hours.
00:16:33.27 Now the actual curling of the root hair, the deformation of the root hair,
00:16:37.21 takes a while to express.
00:16:40.03 Of course we have to keep in mind that this is limited by how
00:16:44.10 fast the root hair can grow.
00:16:46.23 We don't know when the decision to curl is made,
00:16:49.25 but the actual outcome, the mechanics of curling,
00:16:52.26 take somewhat longer.
00:16:54.17 And then, here around 20 hours,
00:16:56.17 we're going to start to get infection.
00:16:58.22 The production of the infection thread and the ability of the bacteria
00:17:02.11 now to penetrate in through that root hair.
00:17:05.13 And then the cell division here leading
00:17:08.24 to the production of the nodule, that's all happening over this
00:17:13.09 period of a day to two days.
00:17:16.00 Now through a set of genetic mutageneses and screens,
00:17:21.18 our group and others have identified many different plant mutants
00:17:25.18 that arrest at one or the other of these stages.
00:17:30.04 And having these subcellular assays to do
00:17:33.02 allowed us to distinguish between the different kinds of
00:17:37.02 plant Nod- mutants. So here's an example.
00:17:41.28 Here, on the left, is a gene called
00:17:46.07 NFP for "Nod Factor perception." If that gene is mutated,
00:17:50.15 you have a block right here, so nothing happens at all,
00:17:54.09 there's no depolarization, there's no calcium flux,
00:17:58.03 no change in the morphology.
00:18:00.24 And then, here are two mutants that are
00:18:06.05 present in such a way that they allow the plant to have calcium flux,
00:18:10.27 and it has suppression of reactive oxygen species and so forth.
00:18:15.10 However, it doesn't show calcium spiking.
00:18:18.00 So these two mutants were able to distinguish
00:18:20.28 between the early calcium flux and the calcium spiking itself.
00:18:25.07 They are not only separated in the cell in geography and in time,
00:18:29.26 but they can be separated genetically.
00:18:33.00 Here, we found one or more mutants that allow calcium spiking
00:18:39.29 but do not allow any gene expression.
00:18:42.22 And so forth and so on down the line.
00:18:45.10 So with all of this, we are able to set a timeline
00:18:49.04 and a developmental sequence for how signals are transduced.
00:18:53.25 And among other things, we were able to use that transcription assay
00:18:58.01 to distinguish between root hair curling genes
00:19:03.19 and early calcium transduction genes.
00:19:06.25 And we were able to show that all of those plant genes
00:19:10.25 that are expressed come on at the same time.
00:19:14.21 It's not the case that you can get a few genes here,
00:19:18.05 and a few more here, and a few more after that.
00:19:20.16 You get no genes expressed at all, up until this point here.
00:19:25.11 And we were able to show that by a combination of
00:19:27.26 mutant analysis and transcription analysis.
00:19:33.07 So that means we're going to be able to say that
00:19:36.29 the series of events of shown here --
00:19:39.25 calcium flux, suppression, growth arrest (the pausing) --
00:19:44.26 can cluster, and we can see that one particular gene called NFP,
00:19:50.09 which we believe encodes a receptor,
00:19:52.09 is responsible and required for those events.
00:19:55.28 Then there's a set of two genes, DMI1 and DMI2,
00:20:00.07 that are required in order to get to calcium spiking.
00:20:03.12 One of these is an ion channel,
00:20:05.13 the other is a putative receptor kinase.
00:20:08.22 Intriguingly, just downstream from calcium spiking
00:20:13.24 is a mutant, DMI3, and another mutant, CYCLOPS;
00:20:17.28 these are both required in order
00:20:20.15 for any of the later events to occur, such as transcription.
00:20:25.09 So we believe that these genes help interpret the
00:20:28.20 calcium spiking signal and transduce it to the
00:20:32.08 next set of genes, NSP1 and NSP2.
00:20:35.04 These are required for transcription, and indeed,
00:20:38.11 they turn out by sequence to be predicted transcription factors.
00:20:42.03 More transcription factors are also required in order for
00:20:45.09 nodulation to occur. Now all of these events
00:20:48.10 on the right require the one pathway, this is called the signaling pathway,
00:20:54.02 starting with the signaling receptor NFP1,
00:20:57.04 and going through transcription factors.
00:20:59.26 But I haven't said anything about infection,
00:21:02.19 and I'd like to mention that.
00:21:05.11 It turns out that the plants appear to have not one receptor, but two.
00:21:10.24 The second receptor is called the "stringent" or "entry" receptor,
00:21:15.11 and the name of that gene is LYK3, in the case of Medicago truncatula.
00:21:20.22 This, and one of the transcription factors,
00:21:23.16 are required in order for infection to occur.
00:21:26.22 So you can have these other events happening, but no infection.
00:21:32.27 This additional set of factors is required for infection.
00:21:38.08 So back to the outline.
00:21:40.12 I've gone through a description of some of the plant cell
00:21:43.17 responses to Nod Factor, and told you about
00:21:45.29 how we have used plant mutants, plus those cell phenotypes,
00:21:49.21 in order to create an ordered set of steps that we believe
00:21:53.06 represents the signal transduction pathway in early nodulation.
00:21:57.28 In the next part of the talk, I'm now going to move, as I say,
00:22:02.13 beyond nodulation: I'll be talking about infection
00:22:05.23 and also about later stages in nodulation.
00:22:10.12 So, as we think about the symbiosis, remember,
00:22:14.26 everything that I described in terms of early signal transduction
00:22:18.24 is over here. That's just the beginning.
00:22:22.19 There's so much more to come.
00:22:24.18 There's as the bacteria infect and eventually form the nodule.
00:22:30.06 So now let's take a look at some of the
00:22:32.10 specific topics that are interesting. First, infection.
00:22:36.13 We've already seen that more genes are needed
00:22:38.26 for infection than just for signaling.
00:22:41.02 And the infection thread is a remarkable structure.
00:22:44.08 It penetrates not just through the root hair,
00:22:47.23 but then through multiple layers of plant cells,
00:22:51.04 on its way in to find target cells.
00:22:56.06 Now, as this proceeds, the plant elaborates an entire nodule,
00:23:02.15 and all the dark cells in this photo are packed full of bacteria.
00:23:08.11 And if you look close up at those,
00:23:10.26 each bacterium is surrounded by an envelope of plant membrane,
00:23:15.24 and within that, the bacteria are going to differentiate.
00:23:19.01 So, using different approaches,
00:23:21.18 we're going to take a look at how the plant manages
00:23:25.19 to support these really remarkable events.
00:23:28.05 We'll start with infection. As I mentioned,
00:23:33.10 infection occurs beginning in the root hairs,
00:23:36.15 and what we wanted to ask is,
00:23:38.00 how does the inside of the plant cell
00:23:40.17 cope with the invasion of the bacterium?
00:23:42.26 How does it reorganize itself?
00:23:44.21 We decided to take a look at some candidate genes called flotillins.
00:23:50.11 These are genes that are originally identified in animals.
00:23:54.02 I'll give a brief review, and then I'll show you
00:23:57.04 our data that demonstrate that plant flotillins are active in nodulation.
00:24:03.22 There's two copies of flotillins that are specifically upregulated
00:24:07.24 in nodules, their transcription is regulated along with other
00:24:13.05 nodulation genes, they're required for infection,
00:24:18.20 they're required for elongation, and remarkably,
00:24:21.28 the proteins of these flotillins have very striking
00:24:25.07 localizations that we believe relate to the
00:24:28.02 mechanism of infection. So, what are flotillins?
00:24:31.06 They've been studied widely in animal cells.
00:24:34.11 This diagram shows an animal cell with plasma membrane.
00:24:38.22 Flotillins are shown here. There's two of them, they're outlined in red.
00:24:44.05 They have a domain that is affiliated with the membrane,
00:24:47.07 and a tail that is more cytoplasmic.
00:24:50.20 And it is thought that they relate to signaling, to endocytosis,
00:24:56.25 and to activity of the actin network within the cell.
00:25:03.26 It's also been demonstrated that these flotillins occur
00:25:10.14 in what are sometimes termed "membrane microdomains,"
00:25:14.16 also sometimes called lipid rafts,
00:25:16.22 although the terminologies are actively being discussed these days.
00:25:24.03 Now plants have flotillins, too,
00:25:25.29 although they have not been widely studied.
00:25:29.03 In Arabidopsis, the sequence shows that
00:25:31.03 there are three flotillins. In our analysis,
00:25:33.27 we found more than seven in the legume Medicago truncatula.
00:25:41.11 Of these seven, we found that two are uniquely expressed in nodules.
00:25:47.01 We found that the proteins are, as with animals,
00:25:50.22 flotillins present in small domains, or puncta.
00:25:55.22 And I'll show you that one of the two has a very special localization
00:26:01.05 during infection.
00:26:02.25 Now, the predicted structures of the flotillins in plants
00:26:06.02 looks very much like the animals',
00:26:07.25 with a head domain and a tail domain,
00:26:11.27 so we might expect that it's going to have some of the same properties.
00:26:16.04 So, as I mentioned, Arabidopsis has only three flotillins,
00:26:20.15 but Medicago truncatula has more than seven,
00:26:24.26 and here's one of the really interesting things for us,
00:26:27.21 is that there are a great number of these that are actually
00:26:31.00 all linked together.
00:26:32.16 Now you'll see here that there's flotillin 3 and 1,
00:26:35.25 then there's down here flotillin 4 and flotillin 2.
00:26:39.00 We believe that those are active flotillins.
00:26:42.03 One of the others, shown here flotillin 5,
00:26:44.07 is in the same genomic regiion, but we believe it's not an active gene.
00:26:51.12 Now, we took a look at the activity of the promoters
00:26:55.16 for each of those flotillins, and what you can see here is
00:26:58.20 that the top, flotillin 1, its promoter is active in the vasculature.
00:27:04.25 Here it's expressing a GUS fusion.
00:27:08.10 Flotillin 3 is also active in the vasculature.
00:27:12.07 But if you take a look at flotillin 2 and flotillin 4,
00:27:15.16 these are being expressed in nodules.
00:27:19.01 We can also quantify the transcript, and the same story emerges.
00:27:23.20 We can see here that flotillin 1 and 3
00:27:26.15 are at a low level throughout nodulation,
00:27:29.09 but both flotillin 2 and flotillin 4 are strongly upregulated
00:27:34.16 in the first day after roots are presented with bacteria.
00:27:40.07 Flotillin 4 comes down, although flotillin 2 stays fairly well expressed
00:27:47.13 during at least a couple of weeks of nodulation.
00:27:51.17 So in terms of their expression,
00:27:54.06 these appear to be especially associated with nodule development.
00:27:59.27 Does that relate to any of the formalities that we know
00:28:03.23 right now about the genetics of early nodulation?
00:28:07.02 So remember that there are a set of genes that I introduced earlier,
00:28:10.28 and we have mutations in these various
00:28:13.23 steps for nodule signal transduction,
00:28:18.26 and that transcription occurs only after a whole set of steps
00:28:24.06 have taken place. So what about flotillin 2 and 4?
00:28:28.20 And as you can see in this bar graph,
00:28:32.02 those two flotillins actually require the same signal transduction pathway
00:28:36.16 in order to be expressed.
00:28:38.08 Here is a wild type on the left, we're seeing flotillin 2 and 4.
00:28:42.17 And then here are a couple of the nodulation mutants.
00:28:47.06 LYK3, NSP2, NIN-1, those are here.
00:28:52.16 NSP and NIN, here's LYK3 for a receptor for Nod Factor.
00:28:58.15 These appear to be required in order for the flotillins to be upregulated.
00:29:03.19 We could also show that some of the downstream genes
00:29:06.09 are not required.
00:29:08.15 Now, we know that these flotillins are controlled
00:29:12.15 in the same way nodulation genes are controlled,
00:29:15.13 but what about their function?
00:29:18.04 Through using RNAi approaches, we can diminish the
00:29:22.05 activity of the various flotillins, and here's one particular experiment,
00:29:28.12 and these results have been borne out in others',
00:29:31.23 though the absolute numbers tend to vary.
00:29:35.11 So if we take a look at a wild-type plant for example,
00:29:38.15 we can see that it's got about six nodules per plant.
00:29:42.06 If you knock out flotillin 1 and 3, there's again about six.
00:29:46.24 But if you knock out flotillin 2, there's many fewer nodules.
00:29:52.02 Flotillin 2 and flotillin 4, if knocked out together, is even more.
00:29:56.21 But flotillin 4 knocked out by itself
00:29:58.23 does not appear to have much an effect on the number of nodules.
00:30:03.04 If you look at the percent of plants with nodules,
00:30:06.08 you don't see a major effect.
00:30:07.28 In fact, if anything, you seem to get a few increases
00:30:12.03 of the percentage of plants that have some kind of growth on them.
00:30:16.16 But now if we look at the percentage of nodules
00:30:20.03 that are actually Fix+, which is indicated by the pink color
00:30:24.29 from leghemoglobin, you can see that about,
00:30:29.05 in this particular experiment, almost 30% of the wild-type nodules
00:30:34.01 appear to be functioning, about the same for flotillin 1 and 3 knockouts.
00:30:40.05 But if flotillin 2 is knocked out,
00:30:43.02 then there's a striking diminution of the number of functional nodules,
00:30:48.24 and a less striking but still significant change for flotillin 4.
00:30:54.16 And the double mutant almost completely wipes out
00:30:58.09 any effectiveness of the nodules that do form.
00:31:01.20 So, we would conclude from that that flotillin 2 and flotillin 4
00:31:05.13 are both required for nodulation, and that they are not redundant,
00:31:09.12 because the double mutant has a more severe phenotype
00:31:12.09 than either of the single mutants alone.
00:31:16.03 We've also now gone to take a look at the proteins,
00:31:18.25 and what we found is the following:
00:31:21.08 Let's begin on the left with flotillin 2.
00:31:24.23 This is a view of epidermal cells,
00:31:26.28 so we're looking down at the surface of the root here.
00:31:29.21 And you can see that flotillin 2 is present in little dots, or puncta,
00:31:34.04 and there's a very striking localization of flotillin 2
00:31:38.17 at the polar end of the cell.
00:31:43.19 In the root hairs, the flotillin 2 is very strikingly punctate,
00:31:47.26 it's present in these little dots, and this kind of distribution
00:31:55.23 remains the case if the plants are inoculated,
00:32:00.04 and that's shown in the lower part of the left-hand side.
00:32:03.13 So, uninoculated and inoculated, flotillin 2 is punctate,
00:32:08.21 it remains fairly stable, although there are some subtle changes
00:32:13.17 in density and in polarity.
00:32:16.23 But we got a very striking result with flotillin 4,
00:32:21.02 and I'll show you that here. Now let's start on the top again.
00:32:23.28 The epidermal cells and root hair cells,
00:32:26.22 and it's very striking that the flotillin 4 signal (a GFP fusion signal)
00:32:32.27 is present in puncta. But even more striking was that,
00:32:37.05 after inoculation, what starts out as an even distribution of flotillin 4
00:32:42.24 in root hairs becomes a highly polar distribution,
00:32:46.29 and that's shown with these red arrows,
00:32:49.11 where you can see that at the tips of these inoculated,
00:32:52.12 bacteria-treated root hairs, flotillin 4 has migrated to the tip.
00:32:58.06 So, that suggests that flotillin 4 has a very active participation
00:33:04.07 in something having to do with what goes on at root hair tips.
00:33:07.13 So we began to look specifically at infection,
00:33:10.21 using wild type as control, where you can see bacteria invading.
00:33:16.07 These blue, lac-stained bacteria show infection threads
00:33:19.29 for bacteria on a wild-type plant.
00:33:24.01 Here's a nice infection thread growing through a root hair
00:33:26.16 on the wild type. But in a flotillin 4 "minus" mutant,
00:33:29.25 things are not working very well.
00:33:32.02 What you're seeing here is that there's no significant
00:33:36.15 penetration deep into the nodule.
00:33:39.20 Furthermore, even if you look at a root hair,
00:33:42.07 the infection threads lack integrity, they're not well structured,
00:33:49.09 and we find in fact that they don't succeed, they don't penetrate.
00:33:54.22 So it appears that flotillin 4 function is important for infection threads,
00:33:59.04 and we also now have evidence that shows that flotillin 4 is
00:34:02.21 associated with infection threads.
00:34:06.12 Flotillin 2 is shown here, bright green fluorescence around the cell.
00:34:13.01 This particular root hair, which is curled, has been infected,
00:34:16.19 and it has bacteria that fluoresce red. Those bacteria are shown here.
00:34:21.04 But you can see that the flotillin 2 is around the membrane of the outer cell,
00:34:26.01 it's not affected, not close by to the red fluorescence of the bacteria.
00:34:31.06 However, if we look at flotillin 4, that's shown here.
00:34:35.12 Flotillin 4-GFP is around the outside, but it's also here, see?
00:34:40.26 Around the infection thread. Green fluorescence,
00:34:44.12 and here's the red bacteria inside the fluorescence.
00:34:47.11 So that suggests that flotillin 4 is affiliated with the
00:34:51.05 infection thread membrane.
00:34:53.02 Now you'll recall that, in the sequence of genes
00:34:57.26 that are important for nodulation signal transduction,
00:35:01.11 one set is called the signaling pathway, here,
00:35:04.11 but more genes are needed in order to get infection,
00:35:07.13 including the entry receptor LYK3.
00:35:10.19 Now that we've got some clue that flotillins are involved in infection,
00:35:14.11 we might want to ask whether we can place flotillins in any kind of
00:35:18.22 relationship to the genes that have been previously found
00:35:21.23 to be important for early signaling and infection.
00:35:24.27 Now, in the following photos,
00:35:27.23 I'm looking at the puncta density of flotillin 4.
00:35:33.06 Here's a wild type, and the puncta density is the number of
00:35:37.19 bright spots per square micron,
00:35:40.28 so you might have a little square micron there,
00:35:45.11 and then you plot the number.
00:35:47.26 And I'm going to compare that wild type to three different mutants.
00:35:52.07 Each of these is a mutant in the putative entry receptor LYK3.
00:35:56.20 lyk3-1, which is the most severe,
00:36:00.21 and two other alleles, lyk3-2 and 3-4.
00:36:06.23 The lyk3-1 is predicted to have a protein with a dead kinase domain,
00:36:13.25 and what we saw looking at this particular index of
00:36:17.07 puncta density is that, if you compare the wild-type number here
00:36:22.12 to the number in the lyk3-1 mutant, the puncta density for FLOT4
00:36:27.01 is greatly diminished, significantly lower.
00:36:30.16 We don't draw any mechanistic conclusion from this,
00:36:33.09 but it did give us the sense that maybe LYK3 itself,
00:36:37.01 together with FLOT4, would be worth looking at.
00:36:41.23 So we want to ask whether LYK3 and flotillin 4 interact.
00:36:46.12 We're going to need to study LYK3,
00:36:48.27 and we were fortunate to collaborate with Doug Cook, Brendan Riely,
00:36:52.22 and their colleagues at UC Davis, and thanks to their contributions,
00:36:57.10 we have the following tools for study:
00:36:59.29 First and most importantly, a stable transgenic of
00:37:03.22 Medicago truncatula that carries a GFP fusion to LYK3,
00:37:07.22 under the control of its own promoter.
00:37:10.00 It's known that this is functional because it complements
00:37:13.06 a lyk3-1 mutant.
00:37:15.19 Because it's under its own promoter, we have more reassurance
00:37:19.00 about its correct position and localization.
00:37:22.17 Now into this stable transgenic, we're now going to introduce
00:37:27.11 a second marker, we're going to create a transformant
00:37:31.02 that also has flotillin 4 linked to the fluorescent mCherry protein,
00:37:37.23 so we'll have green fluorescence for LYK3, red fluorescence for FLOT4.
00:37:41.04 And our particular tool is going to be a spinning-disk confocal
00:37:44.17 at the Carnegie Institution, in collaboration with David Ehrhardt.
00:37:50.04 We were able to visualize the LYK3 putative receptor in root hairs.
00:37:57.08 So the results are shown here.
00:37:59.09 We have LYK3-GFP, it's present in the root hair,
00:38:02.26 you can see that it's punctate.
00:38:07.07 And when we took a look at the LYK3 plant with infections,
00:38:12.14 we found the following: Here's a curled root hair,
00:38:16.02 and you can see going down through the curled root hair
00:38:18.16 is an infection thread, and the green fluorescence of
00:38:21.29 that LYK3 is all the way along the infection thread membrane,
00:38:25.26 so we can see that the LYK3 putative receptor is localizing
00:38:30.07 the same way that the flotillin 4 appears to do along the
00:38:35.12 infection thread, as it moves into the cell.
00:38:38.10 Now, an important control is shown here also.
00:38:41.22 Root hairs and other parts of plant cell walls have
00:38:46.01 autofluorescence as well, and we wanted to ask whether
00:38:50.06 the green fluorescence we saw here was really due to the LYK3,
00:38:54.16 or was it autofluorescence.
00:38:56.09 The results of that are shown in this other panel.
00:38:59.20 Now, this is a fusion of LYK3 to a nonfluorescent protein.
00:39:05.16 And again, there are bacteria that are fluorescing red.
00:39:08.28 Well, what you can see is that it's a good idea
00:39:11.17 to do this control because the crook of the root hair
00:39:15.03 is very highly autofluorescent, so if we were to see this
00:39:19.00 and say it's GFP fluorescence, we'd be wrong,
00:39:22.08 because that's just what root hair cell wall is doing.
00:39:25.23 You can see here the infection thread is moving down
00:39:29.06 with the red fluorescence, and there is no
00:39:34.00 green fluorescence around it.
00:39:35.19 So infection threads are not intrinsically fluorescent;
00:39:39.11 it's only that the LYK3-GFP is providing that signal.
00:39:44.21 So now we know that flotillin 4 and LYK3 appear
00:39:48.16 to localize to the same place.
00:39:50.12 Can we find out anything else about them?
00:39:53.13 So in this, we have started to use that double transgenic.
00:40:01.24 So what's going on here is that this plant has LYK3-GFP.
00:40:06.24 It also has flotillin 4-mCherry.
00:40:09.20 Now, these are closeups below, you can see the LYK3-GFP,
00:40:14.05 flotillin 4-mCherry.
00:40:16.05 And these are uninfected root hairs.
00:40:19.20 And if you merge the two images,
00:40:21.19 it's possible to see here that the green and red are distinct.
00:40:26.00 So that suggests that they are not co-localizing
00:40:29.05 within the distance where their fluorescence would
00:40:32.05 mix and make a yellow color.
00:40:35.11 Now, taking a look at that more statistically,
00:40:38.13 we can do a correlation plot, as shown here,
00:40:41.24 where on one axis, on the x-axis, that's the fluorescence intensity of one,
00:40:47.15 on the y-axis is the fluorescence intensity of the other.
00:40:51.08 And in fact, there's a distribution all the way around,
00:40:55.05 it doesn't particularly correlate.
00:40:57.02 If you have a high fluorescence of mCherry,
00:40:59.19 that could be either low or high fluorescence of GFP.
00:41:04.12 But what happens when we treat root hairs with bacteria?
00:41:10.15 And that's shown here.
00:41:12.12 Now, again we've got the GFP, here's a closeup.
00:41:16.26 We've got the mCherry for the flotillins, and a closeup.
00:41:20.09 But now, when we do the co-localization,
00:41:22.25 you can see that there appears to be a
00:41:25.29 merging of the green and red fluorescence,
00:41:28.25 to present a higher number of puncta that look yellow.
00:41:33.24 So we would say there appears to be an increase
00:41:37.00 in the correlation of their location,
00:41:41.05 and here's another way of looking at that.
00:41:43.22 The correlation plot now shows a correlation coefficient
00:41:48.26 that's more than 0.5.
00:41:52.15 That means that more than half the time,
00:41:55.11 you're getting a correlation of the intensity of fluorescence
00:41:58.04 of one and the other.
00:42:00.08 So that suggests to us that whether or not flotillin 4 and LKY3
00:42:05.13 are associated depends on
00:42:08.13 whether the bacteria have interacted with the root hair.
00:42:11.25 So we pursued that a little more.
00:42:14.03 Another way that we have started to look at that
00:42:16.26 is to examine the dynamics, not just the location,
00:42:20.21 but dynamics of what these proteins are doing.
00:42:23.17 So in this experiment, we're going to be looking at a root hair
00:42:28.22 which has a LYK3-GFP fusion, so that's the only fluorescence
00:42:33.26 you're going to see, it's just the receptor LYK3.
00:42:37.13 And what you're going to be seeing in the micrographs
00:42:40.03 is as if you took a plane like this, right?
00:42:44.20 Through the root hair and, where you see this bright point,
00:42:49.20 that's what is going to be shown now on the films.
00:42:54.22 Now the left-hand...
00:42:56.11 we're going to look at LYK3 in an uninfected root hair.
00:43:01.17 So one of the things you can see is that it's very dynamic.
00:43:05.17 If you try to focus on a point and follow the LYK3,
00:43:10.13 it's moving around too much, you can't do that.
00:43:13.28 But now let's look at a root hair that has been treated with bacteria.
00:43:19.00 What a difference.
00:43:20.10 Now you're looking at the LYK3 fluorescence,
00:43:22.14 and it's really behaving itself. It's sort of staying place,
00:43:25.20 and these two arrowheads, for example,
00:43:27.15 show places that you can focus in,
00:43:29.16 and you can see a LYK3 signal that's not moving, that's fairly stable.
00:43:34.29 So that suggests to us that it's not just where LYK3 is,
00:43:40.07 it's how fast it's moving and perhaps shuttling around,
00:43:45.12 that is being changed by the presence of the bacteria,
00:43:47.24 which is remarkable, and that led us to another way
00:43:52.29 to look at LYK3 together with flotillin 4.
00:43:56.18 In the following, we won't be looking at movies,
00:43:59.05 but we'll be representing the dynamics over time
00:44:01.28 through what's called a kymograph.
00:44:03.20 Now what you're seeing on the left is a micrograph of the receptor
00:44:09.21 LYK3-GFP fluorescence, flotillin 4 fluorescence, and the merge.
00:44:14.24 Likewise, here for bacterially treated root hairs,
00:44:19.16 LYK3-GFP, flotillin 4-mCherry, and then the merge.
00:44:24.22 Now, at each point in time, we don't look at the whole root hair,
00:44:30.16 we're going to just be looking at a transect shown here,
00:44:34.06 and also shown over here, by these blue arrows.
00:44:38.05 And at any one point in time, just that line is going to
00:44:42.13 be represented as follows.
00:44:45.13 Here, if we take this line going across the uninfected LYK3-GFP,
00:44:54.13 then we can see at one 10-second period
00:44:58.11 whether a particular position was light or dark.
00:45:01.23 We can follow that over time and we can see that the correlation
00:45:05.06 is rather loose. However, flotillin 4 is very stable;
00:45:09.11 if you look at the flotillin 4,
00:45:12.07 10 seconds, 20 seconds, and so forth...
00:45:15.02 if it's fluorescent at one 10-second interval,
00:45:18.16 it's highly likely to be fluorescent in the next one,
00:45:20.19 and so you get this set of lines going through.
00:45:24.28 Now, if we take a look at root hairs that are treated with bacteria,
00:45:28.01 we find the following.
00:45:31.23 Again, this is what the root hairs look like,
00:45:36.03 but if we follow them over time, you can now see that the LYK3-GFP
00:45:39.23 is very stable. If that transect shows a bright point
00:45:44.16 at one 10-second interval, it's probably going to be bright
00:45:46.29 the next 10-second interval as well, so the LYK3 has settled down,
00:45:52.02 the flotillin 4 is still very stable, and when you take a look at the merge,
00:45:57.08 you can see that there appears to be a correlation of the brightness.
00:46:02.01 So, as we summarize, we can say the following:
00:46:06.22 That the puncta density is changed for FLOT4-GFP
00:46:14.02 in a genetic background where LYK3 is mutated.
00:46:18.10 We can see that LYK3 itself localizes in puncta
00:46:23.20 that suggests it's in membrane microdomains.
00:46:26.09 Also, it also localizes to infection threads.
00:46:29.25 We found that in uninfected root hairs, the receptor
00:46:35.00 and the flotillin do not have much overlap,
00:46:39.00 and also they are very different in their motility, in their dynamics.
00:46:43.10 However, after inoculation with Sinorhizobium meliloti,
00:46:48.16 the receptor and the flotillin co-localize,
00:46:51.28 and their dynamics remain similar.
00:46:54.26 Not known is whether this is direct or indirect.
00:46:58.01 What is the nature of the protein-protein interactions?
00:47:00.27 Because fluorescence in and of itself is relatively loose,
00:47:06.17 it does not have to have precise molecular adjacency.
00:47:11.29 So further fluorescence studies with FRET
00:47:16.11 and biochemical studies will be necessary,
00:47:18.22 and we think that following the combination of
00:47:22.14 flotillins and receptors is going to be a very exciting way
00:47:26.24 to ask how the plant is mobilizing itself to accept infection.
00:47:31.28 So back to the outline.
00:47:33.22 I've finished talking a little bit about how a candidate gene approach
00:47:38.19 took us from the study of flotillins to the study of infection.
00:47:42.09 In this next segment, I'd like to talk about some mutants
00:47:46.05 that we've isolated called the dnf mutants.
00:47:49.18 These have identified plants genes
00:47:52.09 necessary for the final stages of symbiosis.
00:47:55.24 And I'll talk about two of those, DNF1,
00:47:59.00 which encodes a signal peptidase,
00:48:01.01 and DNF2, a putative phospholipase C.
00:48:05.00 So looking again at the sequence of nodulation,
00:48:07.23 we're now taking a look at an even later stage,
00:48:11.18 where bacteria have penetrated into the nodule,
00:48:15.17 and they get released into the cells, and they're able to fix nitrogen.
00:48:22.11 So the dnf mutants, called "defective in nitrogen fixation,"
00:48:27.04 were isolated out of a screen that we did of
00:48:30.16 mutants that we generated by fast neutron bombardment.
00:48:34.02 This often creates deletions, it's an ionizing radiation,
00:48:39.13 and so these are severe mutants.
00:48:41.28 When we screen them, we found a number of mutants
00:48:45.20 which were like this. Instead of being nice,
00:48:48.12 pink nodules here, the dnf mutants are white,
00:48:51.14 and they're small. They don't have any nitrogenase activity,
00:48:55.21 and that's shown in this slide
00:48:58.13 by assessment of acetylene reduction, shown here.
00:49:02.25 Wild type is able to convert acetylene to ethylene,
00:49:05.21 which an indicator for nitrogenase enzyme.
00:49:08.26 But the dnf mutants, as you can see,
00:49:10.17 have very low or even no ability to fix nitrogen.
00:49:15.22 Now, they also don't seem to have completely normal development.
00:49:20.07 One of the mutants, dnf1, is shown here,
00:49:22.14 and we got two alleles of that, they're very similar.
00:49:25.10 A wild-type nodule has cells filled with bacteria.
00:49:31.14 The bacteria do get infected into the dnf1 nodules as well;
00:49:36.07 nonetheless, they are not as large,
00:49:39.09 and they do not seem to be able to fix nitrogen.
00:49:43.09 So we took a look further at dnf1,
00:49:47.18 and one of the ways in which we studied this was as follows.
00:49:53.23 Here's a wild-type nodule shown in section, and this nodule,
00:49:59.10 in this case, has been established by the action of a bacterium
00:50:04.27 carrying a glucuronidase fusion into the promoter
00:50:08.27 for the nitrogenase gene. That means that if the bacteria
00:50:13.03 are expressing their nitrogenase, then the nodule will turn blue
00:50:18.19 in the presence of a glucuronidase substrate.
00:50:21.09 So, what we see here is that the nifH (which is nitrogenase)-glucuronidase
00:50:26.08 fusion is turned on.
00:50:28.02 That's good, that means that the bacteria are expressing their
00:50:31.15 nitrogen fixation genes.
00:50:33.16 However, what we found with the dnf1 was the following:
00:50:38.14 Taking a look at these nodules with the same exact bacterium
00:50:42.16 carrying a glucuronidase fusion to the nif promoter, we found no activity.
00:50:47.14 You could see here with the red arrow; it's pointing toward the nodule,
00:50:50.14 but there's no blue stain.
00:50:51.22 So we conclude from this that the bacteria are not able to
00:50:55.23 express the genes for nitrogen fixation.
00:50:59.05 So we can say that the bacteria need the plant's DNF1
00:51:03.28 in order for the bacteria to fix nitrogen,
00:51:06.03 or even to express their nitrogen fixation genes.
00:51:09.12 So what is dnf1? This gene in the plant encodes
00:51:14.01 one of the subunits of what turns out to be a nodule-specific
00:51:18.03 signal peptidase.
00:51:19.17 And I'll tell you about nodule-specific
00:51:21.12 and the other components in a moment.
00:51:23.09 But first I'll just review a little bit what signal peptidase is.
00:51:26.18 In a eukaryotic cell, we've got the endoplasmic reticulum,
00:51:31.05 and proteins that are extruded into the lumen
00:51:36.04 may get there by a signal peptide,
00:51:38.27 and if the signal peptidase cleaves off that signal peptide,
00:51:43.29 and that allows the production of the mature protein,
00:51:47.03 which would be shown here.
00:51:48.27 Now, once that mature protein is formed,
00:51:51.25 after its signal peptide is taken off,
00:51:54.11 then it may be targeted for secretion or for vesicle trafficking.
00:52:00.02 So what we found is that DNF1,
00:52:02.22 required for bacterial gene expression and differentiation,
00:52:07.03 has something to do with protein processing.
00:52:10.18 Now let's take a look at the expression of this gene.
00:52:13.10 If you take a look at the vegetative parts of the plant,
00:52:15.25 such as the leaf and stem or flowers,
00:52:20.07 what you find is that expression of the DNF1 signal peptidase subunit
00:52:25.16 is very low, but it's quite high in nodules.
00:52:29.07 It's also present to some extent in developing seeds,
00:52:32.18 although at a much lower level.
00:52:34.22 Taking a look at the time course, what we can see is here.
00:52:37.29 The DNF1, if you plot days of nodule growth and development,
00:52:43.03 and DNF1 expression, you can see that DNF1
00:52:45.18 goes up early in nodule development.
00:52:49.27 You can also track it during seeds and find that
00:52:52.10 it's relatively steady during the maturation of seeds.
00:52:56.12 So, DNF1 is most highly expressed in nodules
00:52:59.11 and it goes up very early in those nodules.
00:53:02.27 Now, let's take a look not just at DNF1, but using transcription,
00:53:09.14 let's take a look at what genes,
00:53:12.06 out of all the microarray data, tend to be expressed
00:53:15.26 whenever DNF1 is expressed, and those are shown here.
00:53:19.05 Now, on the left, you can see the names of the proteins.
00:53:24.27 This is what they do, and what you can see is that there's
00:53:27.09 the other subunits in the signal peptidase complex,
00:53:34.27 are also regulated up in early nodules the same way the DNF1 is.
00:53:40.14 In addition, the signal peptide peptidase,
00:53:42.16 which is needed for completion of the degradation of the signal peptide,
00:53:47.22 that's also upregulated.
00:53:49.23 And finally, this very intriguing protein, SYP132, syntaxin 132...
00:53:55.29 Now what are syntaxins?
00:53:57.27 This is a reminder of how they have been characterized in animal cells;
00:54:02.27 syntaxins are important proteins for vesicle targeting, and in fact,
00:54:10.05 SYP132 here in the plant probably marks the last step of protein secretion,
00:54:16.14 and empirically, it has been observed on plant plasma membranes
00:54:20.29 and also on what's called the symbiosome membrane.
00:54:24.15 That's the membrane that surrounds the bacterium once it's inside the cell.
00:54:28.25 So if we take a look at all of these genes,
00:54:31.04 we can see that they are co-regulated.
00:54:34.04 So we've got a whole signal peptidase complex, and what is it doing?
00:54:38.02 One of the most important questions that could be asked is,
00:54:40.21 what are the substrates?
00:54:42.16 What proteins does this signal peptidase help to mature?
00:54:46.22 And the answer came from work in the labs
00:54:49.23 in Gif-sur-Yvette and in Szeged, Hungary,
00:54:53.24 led by Eva Kondorosi and Peter Mergaert, and their colleagues.
00:54:57.28 And what they have described is a set of proteins
00:55:00.16 called "nodule cysteine-rich peptides."
00:55:04.26 These are characteristic of nodules,
00:55:07.23 they are not expressed anyplace else in the plant.
00:55:10.18 And they are rich in cysteine and bear some resemblance to defensins,
00:55:14.21 which are proteins made by plants and animals
00:55:18.29 in response to microbes.
00:55:21.00 They were able to show using our dnf1 mutant, that one of these,
00:55:26.03 for example, NCR peptide #1, in a wild-type plant,
00:55:31.11 gets processed to a smaller molecular weight.
00:55:35.08 But unless the DNF1 peptidase is there, it does not get processed,
00:55:40.16 because here's our mutant, and you can see that the protein,
00:55:44.13 shown here in immunoblot, is larger.
00:55:47.14 It's because its signal peptide was never taken off.
00:55:50.20 They have also been able to show that, with antibodies,
00:55:54.10 that in wild-type cells, if you use a green dye to indicate the bacteria
00:55:59.19 and a red fluorescence for the antibody to the NCR peptide,
00:56:05.15 they co-localize.
00:56:06.25 Bacteria and NCR peptides co-localize in a normal wild-type cell.
00:56:12.17 Here, they showed an example of what happens in a mutant dnf1 cell.
00:56:18.00 You can see that the bacteria are all over the place in green,
00:56:23.02 but that this NCR peptide,
00:56:26.03 which we know is not being processed, right?
00:56:28.04 Because it's large molecular weight?
00:56:29.22 That NCR peptide is stuck someplace,
00:56:32.17 probably in the endoplasmic reticulum.
00:56:34.25 So, their conclusions, together with our work on the DNF1,
00:56:39.00 are that the NCR peptides are substrates of the DNF1 complex.
00:56:43.17 In wild type, these are delivered to the symbiosome,
00:56:46.07 and in dnf1, they're retained in the ER.
00:56:49.09 They've shown in other work that these mature NCR peptides
00:56:53.12 have profound effects on bacteria,
00:56:55.20 such as causing them to cease cell division
00:56:58.21 and changing their membrane permeability,
00:57:01.23 so that's an ongoing story about the activity of the NCRs.
00:57:06.13 To complete the model so far, we might ask,
00:57:10.20 what does DNF1 do,
00:57:12.11 and one likely activity is that it processes NCR proteins.
00:57:18.23 These end up in the lumen of the ER, and they are targeted,
00:57:22.08 perhaps using SYP132 as part of the mechanism for targeting,
00:57:28.08 into vesicles, which come over and fuse with the symbiosome,
00:57:33.21 in which Rhizobium are going to be differentiating.
00:57:37.22 Now, we also have questions about DNF1 beyond the NCR proteins.
00:57:42.17 For example, DNF1 is present even in legume plants
00:57:46.10 that do not have the NCR proteins.
00:57:49.03 So what is it doing there?
00:57:51.05 Another question is, in the dnf1 mutant,
00:57:57.02 the bacteroids do not differentiate, but they also stop dividing,
00:58:02.05 so it seems that even without DNF1,
00:58:05.05 there may be other signals that are helping these bacteria
00:58:10.03 to stop dividing. So we hope that we'll be able to work on those,
00:58:13.29 as well as on the actual mechanism of DNF1
00:58:17.07 in the next period of our work.
00:58:20.00 I'd now like to finish by telling you about the other mutant, dnf2.
00:58:24.25 And this, as you recall, as shown right there,
00:58:27.27 that also had no nitrogen fixation.
00:58:31.00 And we've done some work to characterize
00:58:34.17 what's going on with dnf2
00:58:36.17 and also to figure out what the gene is.
00:58:38.27 Here is a picture in which you can see,
00:58:41.23 from the root part to the distal part of the nodule,
00:58:46.17 fluorescently labeled bacteria, and they are inside.
00:58:50.08 We can tell that the bacteria get inside the dnf2 mutant nodules.
00:58:56.25 However, they don't express nitrogenase,
00:59:00.03 and they don't express many other bacterial genes either.
00:59:03.29 So, somehow, DNF2 is again required for the bacteria
00:59:08.17 to differentiate and, in fact, to express genes.
00:59:13.24 Now we know that DNF2 is expressed only in nodules,
00:59:18.14 even more strictly than DNF1.
00:59:21.08 This shows you some promoter-GUS fusions
00:59:23.25 where you can see that the early part of the nodule is expressing DNF2,
00:59:29.23 and here you can see it at the tip,
00:59:31.15 and then it diminishes just slightly as the nodules get somewhat older.
00:59:38.19 What is DNF2?
00:59:41.01 It appears to be similar to a phospholipase C,
00:59:46.07 but it's not a canonical phospholipase C the way we're used to seeing.
00:59:50.21 Here would be the classic PLC.
00:59:53.01 You can see it's got the inositol trisphosphate binding, EF-hands,
00:59:59.29 catalytic domains, calcium-binding domains, and so forth.
01:00:04.16 The DNF2 is only homologous to
01:00:09.13 one part of this large, complex phospholipase C.
01:00:15.03 However, we do find that it is very, very similar
01:00:19.01 to a phospholipase C whose model is shown here.
01:00:23.27 This is actually a bacterial phospholipase C,
01:00:27.01 and if you take this in red and then superpose DNF2,
01:00:31.21 it does appear to be very, very similar in its 3D structure.
01:00:36.13 So we want to know what's the biochemical activity;
01:00:39.21 sequence alone doesn't tell us whether it really acts
01:00:43.06 to process phospholipids or as a signaling.
01:00:50.08 But we found one other thing that's really surprising to us.
01:00:54.04 First, as I mentioned, it's only expressed in nodules,
01:00:58.14 but secondly, it's not expressed in the cells of the plant that have Rhizobium.
01:01:05.25 It's expressed in the noninfected cells of the nodule.
01:01:09.08 And that's really a first.
01:01:11.26 There are known metabolic enzymes in soybean, for example,
01:01:15.24 that are present in uninfected cells.
01:01:18.04 But there has never been found a regulator
01:01:20.20 that appears to be active in the noninfected cells of the root nodule.
01:01:26.08 So, what we're trying to ask ourselves is,
01:01:29.20 does the DNF2 protein mediate the infection process
01:01:33.21 directly or indirectly?
01:01:36.02 Is it acting in some way that allows the noninfected cells
01:01:40.08 to send signals or perhaps to avoid signals?
01:01:43.25 So those are some of the questions that lie ahead for us.
01:01:47.21 So winding up the combination of the studies that I've shown you today,
01:01:51.27 I think that the future of our field is going to be very exciting
01:01:54.28 as we take the results of the plant mutants and
01:01:58.03 we study the proteins that are defined by those mutations,
01:02:01.26 and look at how those work, both plant and bacteria together,
01:02:05.24 not looking just at position, but looking at time.
01:02:08.24 We're interested in the great questions of:
01:02:12.29 What the signals are,
01:02:14.15 and how the signals are transduced.
01:02:16.19 Given that so much complicated cell biology has to happen
01:02:20.19 between the beginning of the infection thread and the maturation of the symbiosome,
01:02:25.02 we want to know not just the regulation,
01:02:27.10 but what is the machinery of this infection,
01:02:30.06 and we believe that both the proteins and molecular probes
01:02:33.27 that we can obtain can be used to study the individual steps of
01:02:38.02 nodulation as they move forward.
01:02:41.04 So that concludes our journey through the plant side of symbiosis.

Videos in this Talk
  • Part 1: Symbiosis between nitrogen fixing bacteria and legumes
    Part 1: Symbiosis between nitrogen fixing bacteria and legumes
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 2: Function and regulation of Sinorhizobium nodulation genes
    Part 2: Function and regulation of Sinorhizobium nodulation genes
    Audience:
    • Researcher
    • Educators of Adv. Undergrad / Grad
  • Part 3: Plant genes and cell response in nitrogen-fixing symbiosis
    Part 3: Plant genes and cell response in nitrogen-fixing symbiosis
    Audience:
    • Researcher
    • Educators of Adv. Undergrad / Grad
Speaker: Sharon Long
Total Duration: 2:20:23
Recorded: November 2010
All Talks in Plant Biology

Talk Overview

Legume plants form specialized root nodules to host “rhizobia,” nitrogen fixing bacteria. Rhizobia hosting legumes are able to grow without exogenous nitrogen fertilizer allowing them to be high in protein and to provide nutrition to surrounding plants. In part 1 of her talk, Long gives an overview rhizobium-legume symbiosis including infection and nodule formation and the chemical signals exchanged between the plant and the bacteria.

In Part 2. Long goes into more detail on how chemicals released from legumes increase transcription of specific bacterial genes required for plant infection, while in Part 3 she describes how bacterial factors induce production of specific proteins by the plant. By using a custom gene chip, Long can follow the regulation of both Sinorhizobium genes and alfalfa genes at the same time, during nodule formation. This has allowed her to show that this is an extremely complex process with the expression of hundreds of plant and bacterial genes changing during this process.

Speaker Bio

Sharon Long

Sharon Long

Sharon Long received her undergraduate degree from California Institute of Technology and her doctorate from Yale University. She has been a professor of Biological Sciences at Stanford University since 1982 and she was Dean of the School of Humanities and Sciences from 2001-2007. In her research, Dr. Long employs a spectrum of approaches including microbial… Continue Reading

Playlist: Symbiosis

Related Resources

  • Gibson KE, Kobayashi H, Walker GC. 2008. Molecular determinants of a symbiotic chronic infection. Annu Rev Genet. 42:413-41.
  • Giles E.D. Oldroyd and J. Allan Downie. 2008. Coordinating Nodule Morphogenesis with Rhizobial Infection in Legumes. Annual Review of Plant Biology 59: 519-546.
  • Keith Roberts. 2007. Handbook of Plant Science, Volume 1.

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