Plant Virus Infection and Crops

Part 1: Biology of Plant Virus Infection

00:00:04.00 Good morning, thanks for joining the session this morning.
00:00:06.26 I'm Roger Beachy, a plant virologist and I work in biotechnology as well,
00:00:12.03 I'm from the Donald Danforth Plant Science Center in St. Louis, Missouri,
00:00:15.25 we're a new, relatively new, non-for-profit research institution,
00:00:20.05 and I want to talk to you today about viruses and plants.
00:00:24.04 I'm going to tell you a little bit about how they replicate, but more importantly,
00:00:27.12 the cell biology of how they set up the virus replication factories
00:00:32.00 and how the ability of the virus to use host mechanisms to move from cell to cell eventually ends up in pathogenesis.
00:00:39.27 The second part of my lecture, the second lecture,
00:00:42.21 will be related to how to use that information through biotechnology to develop virus-resistant crops.
00:00:48.25 So, I want to tie these two together, and hopefully what I introduce you today, in this first lecture,
00:00:54.01 will help you in the second.
00:00:56.00 So, what I want to talk about, again, is the cell and molecular biology of virus infection, replication and pathogenicity.
00:01:03.15 This slide shows several examples of virus infected plants.
00:01:08.13 The disease that's shown here results after the virus infects perhaps a single cell, or multiple cells,
00:01:15.00 spreads from cell to cell and then moves from one leaf to the next,
00:01:19.06 eventually invading the whole plant.
00:01:22.01 And as you see, the examples I have here are for cassava, in Africa, in papaya, throughout the world,
00:01:28.20 and this is an example of a disease on a tomato plant that shows severely affected plants in the front,
00:01:35.07 and disease resistant plants in the back.
00:01:38.00 Again, the result here is a culmination of events that leads from infection of a single cell
00:01:45.00 and spreading throughout the leaf and then throughout the plant.
00:01:48.18 Throughout my lecture, I'm going to talk primarily about tobacco mosaic virus.
00:01:52.07 It's a very simple plant virus, it's been used as a model for more than fifty years,
00:01:56.25 and scientists throughout the world have used it for a variety of studies.
00:02:00.18 I'm going to talk about it in terms of its cell biology and molecular biology of replication and spread.
00:02:06.19 There are lots of different examples of viruses in this category,
00:02:09.29 some in fact have hosts like tomato and peppers, but others have been identified that infect Arabidopsis and other model plants.
00:02:18.13 A little bit of a background.
00:02:21.03 The genome of tobacco mosaic virus is a single, very simple RNA molecule.
00:02:26.26 It encodes three genes. The first is an enzyme that helps the virus copy itself, it's an RNA dependent RNA polymerase.
00:02:35.22 The green box indicates the protein that's encoded by the virus to help it to move from cell to cell,
00:02:42.16 and the blue box indicates the location of the capsid protein.
00:02:46.28 The capsid, of course, its role is to replicate, is to encapsulate the single-stranded genome
00:02:53.18 and allow it to be transmitted from cell to cell, or from leaf to leaf,
00:02:58.03 and of course, that's what's transmitted as workers in the field propagate plants and move the virus around.
00:03:04.19 Now from the standpoint of molecular virology, this is a slide from Dr. Milton Zaitlin at Cornell University,
00:03:12.07 and you see that in the first iteration, a virus enters the cell, usually by a wound,
00:03:18.23 either by an insect or a worker who might be working in the field,
00:03:22.07 transmitting the virus into a broken cell, and from there on, the molecular structures take over,
00:03:29.13 and processes take over, the virus is encoded, releasing the viral RNA, ribosomes translate it,
00:03:34.21 and those enzymes that are produced from the translation are responsible for copying the RNA
00:03:40.11 and building the virus replication factories to make more virions,
00:03:44.22 eventually ending up with more virus particles that are shown down there, down in the lower left hand corner.
00:03:51.09 Now, from a cell biologist's point of view, it's a very different story.
00:03:55.24 In this case, we've looked at how the virus enters a cell, and in the first iteration, the virion is dis-encapsidated
00:04:05.17 within a few seconds after it enters a cell.
00:04:09.09 Viral RNA is then moved into, on ribosomes, it's moved into the membranes that are perinuclear
00:04:15.13 and there it begins its translation and set up the first parts of the protein machinery that are necessary to produce more virions,
00:04:22.18 then moves, those membranes then move out into the cytosolic portion of the cell,
00:04:28.00 and set up larger factories, and continue to build them on endoplasmic reticula,
00:04:33.01 until it makes very, very large bodies.
00:04:36.00 Those bodies, then, reproduce themselves, move around the cells, as I'll show you later,
00:04:41.07 and eventually, then move from cell to cell.
00:04:43.23 And the real challenge here is for the virus to use the cellular machinery to do all of these processes
00:04:50.24 and to move from cell to cell.
00:04:52.11 Unlike animal viruses, plant viruses can't bud from their cell membrane and then be adsorbed by others
00:04:59.15 because plant cells are surrounded by very rigid cell walls.
00:05:03.22 These cell walls are penetrated by structures that are known as plasmodesmata.
00:05:09.12 This cartoon, developed by my colleague Bernie Epel, in Israel, shows a little bit of the structure of those plasmodesmata.
00:05:21.19 This represents a cell wall between two adjacent cells, that you see here and over here.
00:05:28.03 And going through that hole in the wall is endoplasmic reticulum.
00:05:34.09 Now, myosins and actins and other energy conducting structures
00:05:42.06 make modifications throughout the living part of the plant's life cycle
00:05:48.02 and allows this membrane to move from one cell to the next in a very metered way.
00:05:55.15 These structures maintain homeostasis. They're very small and only allow very small molecules to move through,
00:06:01.28 including ions and energy carrying materials.
00:06:04.24 If that's disrupted, of course, that changes the homeostatic nature of the leaf and it would be damaging.
00:06:12.04 There are in fact, during early parts of development and later parts of development,
00:06:17.07 you can see some changes in the structure of these plasmodesmata.
00:06:21.17 Here you see they're very much more rigid and they're bolstered by additional beta-1,3-glucans
00:06:27.25 and other molecules that give some rigidity to those structures.
00:06:30.11 So, the question is, how does a virus manage to squeeze through that structure?
00:06:35.05 And different viruses do it in different ways.
00:06:37.23 In this diagram, we've shown a diagram for how a set of viruses called geminiviruses
00:06:45.16 move from cell to cell. These are single-stranded DNA viruses, they replicate in the nucleus,
00:06:50.17 they come out to the cytoplasm, and one of the parts of the protein-coding encoded by the virus
00:06:57.25 moves it from the nucleus to the cytosol, and then another protein carries it all the way through the plasmodesmata,
00:07:03.26 as you see over here on the side.
00:07:05.05 Tobacco mosaic virus, by contrast, does it in a very different way.
00:07:10.01 There's an interaction between the movement protein, membranes and the cytoskeleton,
00:07:15.02 which carry these structures to, and through, the plasmodesmata.
00:07:18.12 And that's a subject of most of my talk this morning.
00:07:22.08 What we've done is use tobacco mosaic virus in a variety of ways.
00:07:27.01 We of course use the fluorescent protein green fluorescent protein as fusions to the movement protein,
00:07:32.26 sometimes as fusions to the code protein, to follow infection around the cell, and between cells.
00:07:39.00 In other cases, we've used simple confocal microscopy, coupled with immuno-localization assays, or procedures.
00:07:49.08 So, let's get into the nature of infection.
00:07:52.24 In this structure, in this study we've infected the virus into a protoplast,
00:08:00.14 these are cells from which we've taken the cell wall, and so that's why you see it as a round structure,
00:08:06.27 and we've identified the three different virus proteins with different colors.
00:08:12.22 The red represents the location of the virus replicase, using an antibody specific for the replicase.
00:08:19.06 The green represents the location of the movement protein, and the blue, the site of the coat protein.
00:08:28.08 Now, this is at about fourteen hours post-infection, and as that structure rotated,
00:08:32.26 you were able to see that some of these spots overlay each other.
00:08:36.20 What that should indicate to you is that they're in similar locations.
00:08:40.16 But as that went around, you know that at fourteen hours,
00:08:43.16 most of those structures were on the surface of the protoplast, on the outside.
00:08:48.10 In this case, now at 21 hours post-infection, now as you see this rotation,
00:08:53.10 you see that the green bodies are larger than they were in the first slide that I showed
00:08:58.01 and you see a little bit more overlap between the red and the green
00:09:01.18 and we'll come back to that a little bit later, that would indicate a co-localization of the replicase and the movement protein.
00:09:08.04 Notice that the structures that are green are much larger in this slide then they were in the previous.
00:09:14.00 This is at 21 hours after infection.
00:09:16.19 Now, a few hours later, at 26 hours post-infection, you see these very large green masses.
00:09:22.18 These are essentially the very large virus replication complexes, or VRCs, as we've called them throughout this talk.
00:09:32.13 And note again that there's a lot of overlap between the green and the red,
00:09:37.05 and the blue color, where the virions are assembled, tends to lie around the outside of these complexes.
00:09:43.27 So, from a standpoint of light microscopy, it's pretty clear that these are large structures that take over a large part of the cell.
00:09:51.28 It's interesting to note that in this infection, the cell can produce as many as ten to the six virus particles per cell,
00:09:58.15 but does not lyse the cell. So, it's learned to live in harmony with the cell by sequestering its replication away from other parts,
00:10:07.22 allowing the cell to continue its normal metabolism and function.
00:10:12.04 We're looking at this now in a relatively early stage of infection,
00:10:17.13 but now cutting through the sections of these immuno-localized proteins.
00:10:23.11 And you see that you see a lot of green, again, the movement protein, located around the outside of the cell,
00:10:29.11 in these large replication factories, and in red, notice,
00:10:33.22 what surprised us in some of these studies, what in some cases you see mostly replicase in one section.
00:10:39.24 In other cases, you find the red and the green overlapping each other.
00:10:43.19 It's clear that the red is inside and the green is on the outside, as shown on this slide.
00:10:49.23 You see at the one over to the right hand side of the slide, it's easy to see the red on the inside and the green on the out.
00:10:57.19 And note that the red is sort of contiguous in some of these photographs.
00:11:02.13 For example, in this one over here, you see the red that goes all around that body.
00:11:07.02 We take that to mean that the red is on endoplasmic reticulum, which would of course have a lumen,
00:11:12.07 and the green would represent the movement protein, which is over top of the replicase.
00:11:17.10 The movement protein also itself interacts with the membranes in some cases, and in some ways.
00:11:24.10 So, what we've struggled with is understanding how the movement protein,
00:11:27.15 which is responsible for helping the virus move from cell to cell,
00:11:30.21 functions in building these replication factories, if at all.
00:11:34.17 And if the function in fact is to move it from the site of where these factories are located
00:11:39.26 over to and through these plasmodesmata, we're curious what the architecture of that structure is,
00:11:46.00 and we'll come back to that a bit later in the talk.
00:11:48.06 So, here's a thin section micrograph view of the virus replication complexes.
00:11:55.09 The arrows outline a series of structures that you see here, that's our interpretation of a virus replication complex
00:12:05.08 that you saw in a previous slide, as indicated by fluorescence microscopy in the confocal image.
00:12:11.03 Now, Dr. Katherine Esau more than fifty years ago described these rope-like structures that are found inside this complex.
00:12:18.07 Notice that there is not an indication that this complex is surrounded by membranes.
00:12:23.09 In fact, there's no indication at all that there are membranes except on the inside of the complex.
00:12:28.06 So then one wonders, how does a cell manage to wall off this thing so it doesn't take over the cell?
00:12:34.07 Or, how does a cell, how does a virus complex manage to escape the degradative enzymes that are present in the cytoplasm?
00:12:42.09 The RNases and the proteases, what keeps this all working together so that it makes more virions,
00:12:47.29 where you don't have disruption of either the virus replication complex, or severe disruption of the cytosol,
00:12:53.22 and killing of the cell.
00:12:55.16 It's especially interesting when one considers silencing of RNAs
00:13:00.17 and how the cell can defend itself against intruding pathogens, such as viruses,
00:13:06.00 how does this manage to keep out the silencing mechanism so that it doesn't degrade the pathogen in the process.
00:13:13.13 In fact, the host has very little defense against this kind of virus.
00:13:18.07 This is now a close-up view of these structures, and notice these rope-like structures.
00:13:22.22 So, what are they?
00:13:24.07 And there are, if you look carefully, there are other membranes.
00:13:27.03 And more recently, we have used immuno-gold localization to locate the proteins
00:13:32.12 and sure enough, on these structures, on these rope-like structures, one finds virus replicase and some movement protein.
00:13:39.14 But what's the structure of them?
00:13:41.07 And recently, we've worked with colleagues at the University of Chicago
00:13:44.09 to begin to tear this apart by using tomography.
00:13:48.09 And I don't have all the data are not in yet, but eventually,
00:13:51.22 as you know, a tomogram is developed on relatively thick sections that are produced for electron microscopy,
00:13:59.02 in the standard fashion, and then uses a high energy beam to itself slice farther down into that section
00:14:07.15 than is seem in normal transmission electron microscopy.
00:14:11.11 And then the section is tilted very slightly, at one or two degrees at each time,
00:14:17.23 and then a new photograph is taken.
00:14:19.20 And from that, one builds an image. Now, we don't have the work finished yet,
00:14:23.07 but I wanted to tell you where we are to date.
00:14:25.27 So, we have been working towards creating a tomogram
00:14:29.12 and of course, using these tilting stage and collecting vast amounts of information,
00:14:36.05 we then compile that information into a three dimensional image that tells us more about how the structure works.
00:14:43.10 And this is just a preliminary, but you can see where we're headed.
00:14:47.12 Note these rope-like structures that you see here; that's like what I showed you before,
00:14:53.09 but since this is now a tomogram, and it's a non-stained section, it's a little less distinct.
00:15:00.00 But you can see, over towards the middle of this slide,
00:15:03.14 this continuous area that we have outlined in a very light green color,
00:15:08.26 and you see that's what we represent as the endoplasmic reticulum.
00:15:13.15 So, inside of this lies the lumen and on the outside, this is the outside structure of the endoplasmic reticulum.
00:15:20.10 Now, if you recall your cell biology, there are rough endoplasmic reticula and smooth endoplasmic reticula
00:15:26.20 and in fact, these represent ribosomes on this one, so this would be a rough ER.
00:15:32.07 When I was a post-doc nearly 35 years ago, I was curious about how viruses are assembled
00:15:37.18 on these membranes and finally, we were able to see it using the new tools of high energy electron microscopy and tomography.
00:15:46.10 Now, the structures that are indicated here in this other color, kind of a magenta color,
00:15:51.25 are ribosomes that link the ER and the rope-like structures.
00:15:58.22 Now, if we were guessing, and we are simply guessing right now,
00:16:03.16 we think that these are the links that hold the ER and the replicase and the ropes all together.
00:16:11.01 And they're in a structure that, with this linkage of rough endoplasmic reticulum and poly-ribosomes and replication,
00:16:19.14 all happening on the same complex that builds into this rope-like structure.
00:16:23.13 We hope over the next several years to be able to tell you more about these complexes.
00:16:28.18 They really have given us an insight as to how these complexes are built.
00:16:33.12 That's one side of the challenge.
00:16:34.21 The next side of the challenge is how does this thing move around the cell and between cells,
00:16:39.03 if in fact it does.
00:16:40.25 Now, what we see here is an image of an infected plant leaf.
00:16:47.17 Now, this is a transgenic plant in which the actin is labeled also with talin.
00:16:54.11 So, you can see the fluorescence of the actin cytoskeleton.
00:16:59.05 Up here, of course, you can see the location of a stomata, the holes through which oxygen enters,
00:17:07.12 and gaseous exchange occurs.
00:17:10.01 Here you see a non-infected cell. And down in this section you can see in fact that there are these globs,
00:17:17.24 these blobs that I talked about earlier.
00:17:20.17 Those represent the virus replication complexes that we talked about before.
00:17:24.20 So, what we're looking at is a very interface between an infected cell and a non-infected cell,
00:17:31.20 this one being non-infected,
00:17:33.12 and these down here being infected.
00:17:35.10 Notice that there is not, in addition to having these blobs, or virus replication complexes
00:17:42.15 in the cytosol, there are also some that are tightly oppressed to the cell wall.
00:17:46.29 The position near the cell wall represents where the plasmodesmata are.
00:17:51.11 That we know by electron, other electron micrographic studies.
00:17:54.25 And so this front shows that in this cell, at this point, is not heavily infected
00:18:02.03 but yet there are still some plasmodesmata that are labeled with the movement protein
00:18:09.00 and the replication complexes, either in or adjacent to the cell wall.
00:18:14.05 In other cases, you can see that these factories are still out in the cytoplasm.
00:18:18.05 This would represent where most of the virus replication is happening early on,
00:18:22.09 or at least where most of the movement protein is being made,
00:18:24.19 and then in this cell, it's a younger infection and this cell is a non-infected cell.
00:18:29.28 Now, if we put this into some sort of imaging, into a time frame,
00:18:36.08 you can see that these globs move around within the cell,
00:18:39.04 and they fuse together, you saw that one just fuse with the large one, the small one fuse to the large one.
00:18:43.27 This large guy is going to start moving down on these actin cables as well and moves all around the cell.
00:18:50.00 That movement is very dynamic and we can mark it by simply putting the microscope on a cell
00:18:58.05 and plot the rate at which it moves by simply measuring the rate of movement in real time.
00:19:05.19 So, in the next slide, I'm going to show you a series of stages of infection.
00:19:12.01 One will be at 18 hours, at early after infection, 14 hours, and then 16 hours and 18 hours.
00:19:17.26 I want you to look at the 14 hour infected cell and you'll see that the bodies are very, very actively moving around the cell.
00:19:25.24 And then the 16 hour stage, the bodies are moving very, very slowly.
00:19:31.01 By 18 hours, they're dead stopped.
00:19:32.28 And then nothing happens for a couple of hours and then I'll show you, down in a lower portion of the slide,
00:19:40.01 a section in which the virus infection has moved from one cell to the next.
00:19:45.08 So, again, up in the upper left-hand corner, that's where the infection is now only 14 hours old
00:19:52.22 and you see those bodies are moving very, very rapidly, as indicated by the number below, 116 nanometers per second.
00:19:59.29 At 16 hours after infection, those bodies are moving more slowly.
00:20:04.06 Very slowly indeed.
00:20:05.10 By 18 hours, they've essentially stopped.
00:20:08.09 And then at 20 hours, two hours later, you now see the infection is not just in a single cell,
00:20:13.05 but is in three cells. The virus infection has moved from the first cell out to the next.
00:20:17.21 And now the cells that are recently infected, those that are up here in the one portion
00:20:23.26 and those that are down in the lower portion, are moving very rapidly.
00:20:27.21 They're in fact moving at the same rate as the bodies at the 14 hour infection.
00:20:33.04 What that told us was that in the first infected cell, it takes a long time to set up the virus replication factories.
00:20:41.17 They start, they're made in that first 6 to 12 to 14 hours,
00:20:45.26 by 14 hours, they begin to move around and fusing with each other and moving to the cell walls.
00:20:51.03 And then they stop moving, there's an immobility to the whole, to the whole process,
00:20:57.06 we had noted this before, that there's a time frame in which the mobility of cytoplasmic bodies stops.
00:21:06.08 At this point, these, you have the replication factors against the cell wall
00:21:10.27 and then we have other films that show the virus bodies, these replication complexes moving from one cell to the next.
00:21:19.19 That surprised us.
00:21:20.28 It was the first indication that what was moving from cell to cell was not the virion,
00:21:25.08 but some sort of a pre-virion complex,
00:21:27.16 perhaps containing viral RNA, maybe even double-stranded RNA,
00:21:31.21 and replicase and other components of the factory that were necessary to get it all started again.
00:21:39.00 Then it goes into the next cell
00:21:40.14 and the replication cycle doesn't have to start from zero.
00:21:43.25 It now can start from having built up, from that first complex that was sent over.
00:21:48.04 In fact, by, if the first infection requires 20 hours to move from the first cell to the next,
00:21:54.11 going from cell number two to cell number three to cell number 4 is about an interval of four hours each.
00:22:00.16 So, it moves very rapidly from cell 2 to cell 3 and that's why the virus infection can move throughout the cell,
00:22:06.25 and throughout the tissue, throughout the whole plant, in a relatively short period of time.
00:22:11.17 It moves between cells, not as virions, but as sort of pro-virions, or pro-structures that include the replication complex.
00:22:21.19 What holds all this together?
00:22:23.27 We've been looking at the structure of the movement protein and asking how it can work.
00:22:29.20 It appears to be an integral membrane protein.
00:22:33.10 It has a trans membrane domain that's fixed here,
00:22:38.09 if we make mutations in those amino acids that are in the membrane portion, as indicated by the red bubbles,
00:22:43.27 we changed the localization and it no longer integrated into the membrane.
00:22:50.02 We do not have a structure of the movement protein
00:22:52.16 but this is a good representation, at least where we are today.
00:22:56.00 On the cytoplasmic side, as you see up here,
00:22:59.23 these are the cytoplasmic side where both the N-terminal and the C-terminal parts of the protein are exposed,
00:23:05.20 there's also a myosin binding site that a former colleague has identified.
00:23:10.24 Those, that might indicate that by binding here, in this region, with the myosin and linking to actin
00:23:20.21 begin to, helps to tell us how these structures move around and through the cell.
00:23:25.23 After these years, and we've been looking at this for a number of years,
00:23:29.15 we have the following picture of how tobacco mosaic virus as an example of a plant virus moves from cell to cell.
00:23:37.03 In the early stages of infection, as I indicated earlier,
00:23:40.21 the virus encodes these proteins that are replicase and movement protein
00:23:46.24 and in some cases, we can see that they go to different sub-sections of the membrane structure of the cell.
00:23:55.01 Those then collapse back onto the nucleus, where the first set of the replication happens.
00:24:01.21 It then moves out into the cytoplasm and builds large replication factories,
00:24:05.29 some of which are shown here in these big globs.
00:24:09.04 Some of these, but not all, will bind to actin-myosin filaments,
00:24:13.26 and then move around the cell. In that process, many will move over adjacent to the plasmodesmata.
00:24:21.09 Now, the question remains how these bodies might move from here to here,
00:24:26.18 a colleague Bernie Epel in Israel has indicated that that movement is driven by a variety of motive forces
00:24:35.13 which help to move proteins between cells. It's a very curious and interesting process.
00:24:40.00 But yet, one has to imagine that the architecture of these walls also change.
00:24:46.08 Now, these are very rigid cell walls, so there must be some softening of the walls,
00:24:50.14 with enzymes that are carried perhaps on the same complexes that create the virus replication complexes,
00:24:56.02 extruding those into this space between the membrane and the cell wall, softening it to the point that it opens and closes.
00:25:03.09 Working with a colleague, Karl Oparka, nearly ten years ago, we found that these holes,
00:25:11.21 these plasmodesmata open for a short period of time at the very leading edge of the infection site
00:25:17.29 on the inoculated leaf, or the infected leaf.
00:25:20.29 So, it opens and then allows this to happen, to transmit, and then the hole closes again.
00:25:27.17 That's in line with the importance of maintaining a homeostatic situation within and between the cells.
00:25:33.29 So, that's where we are in the model.
00:25:35.23 There are lots of questions that remain and we and others have been looking at it,
00:25:40.21 though it's not an easy, the questions are not easy to address.
00:25:45.23 For example, how does a replicase work with a movement protein in the ER, in interactions that create the virus replication complexes,
00:25:55.28 and move them around the cell.
00:25:57.16 What are precisely the interactions that we're going to, hopefully we're going to see some of those in our tomograms that we're currently working on.
00:26:05.13 Certainly there have to be host proteins involved and from a virology standpoint,
00:26:10.26 one suggests that the sites on the endoplasmic reticulum are kind of internal receptors,
00:26:17.17 the places inside the cell where virus proteins go, where they know where to locate,
00:26:22.13 perhaps because the nature of the membrane itself or because of proteins that are embedded in the membrane.
00:26:27.05 The nature of those will take a proteomics approach for analysis and then some genetics to try to interfere with the process of docking.
00:26:37.14 Once we know more about those structures to which the replicase and the movement protein dock,
00:26:41.23 we'll know more about these internal cellular receptors, perhaps they can then be targets for blocking
00:26:46.24 or they will tell us more about the assembly of these factories and then that will tell us how they move around the cell.
00:26:53.12 How they attach? We don't know.
00:26:56.15 And then how do these attach to the myosin-actin fibers for transport to and through the plasmodesmata,
00:27:02.29 what's the process of opening and closing, and essentially, how does a cell manage to live through this process,
00:27:09.28 where the structure is re-oriented and reorganized, making perhaps as many as ten million virus particles per cell, and not killing the cell.
00:27:19.15 What is this compatibility that happens in pathogenicity for this virus?,
00:27:23.11 are some of the questions that we're addressing.
00:27:26.07 In the subsequent lecture, I'm going to talk about how we use this information to develop genetic strategies,
00:27:33.10 or genetic engineering strategies, to help the plant block some of these processes
00:27:38.05 and then thereby developing virus-resistant plants.
00:27:41.20 Some of these are crop plants that will be useful for producing food in agriculture settings.
00:27:49.01 So, I hope you'll join me for the second lecture and I'll sign off for now and leave you with these questions.
00:27:53.03 Perhaps you will be the one that will answer what some of these questions in the future. Thanks.

Part 2: Genetic Engineering for Virus Resistance in Plants: Different Viruses Demand Different Strategies

00:00:03.04 Hi and welcome to the lecture. I'm Roger Beachy,
00:00:05.23 the president of the Donald Danforth plant science center in St. Louis, Missouri.
00:00:09.06 Some of you saw the previous lecture in which I talked about the replication and the spread cell to cell
00:00:17.11 of tobacco mosaic virus, a model virus.
00:00:21.12 In this lecture, I want to talk about how we have used that information
00:00:26.12 and previous information to develop strategies for transgenic plants
00:00:33.05 that would be resistant to virus infection.
00:00:35.27 I'm going to show you several examples and the bottom line, I guess the take-home message is
00:00:40.17 first, if you're going to do the studies that are hopefully have applications in biotechnology,
00:00:47.13 you need to know your pathogen, know how it works, how it's transmitted from cell to cell,
00:00:51.24 how it's transmitted from one plant to the next, so that you can use genetic intradictions, or gene therapy approaches
00:00:58.28 to improve the resistance of plants to infection or replication or a spread,
00:01:04.14 and therefore reduce the impact of disease in a plant.
00:01:09.16 We're going to talk about several viruses in this talk
00:01:13.08 and first I'm going to review some of the strategies that have been used for developing virus-resistant plants.
00:01:19.25 I'll talk about tobacco mosaic virus as a model of how we use this information to develop what we call coat-protein mediated resistance
00:01:28.13 and a real live example of how we've applied this, or are applying it now,
00:01:32.15 in combination or collaboration with our colleagues in India,
00:01:37.13 to develop resistance against a very severe disease in groundnuts, or peanuts,
00:01:42.25 a crop which affects many small holder farmers in India.
00:01:48.03 If we can solve this problem, we will be very pleased of course.
00:01:51.25 And then I want to talk about a very different example, rice tungro bacilliform virus, it's a pararetrovirus
00:01:57.11 and you'll see the strategies that we used here are very different than the ones that we used up above in tobacco mosaic virus.
00:02:02.25 And lastly, another challenge that a colleague of mine, Claude Fauquet, and his lab are working on:
00:02:08.08 so, single stranded DNA virus, a very different type, and the strategies used there will be different
00:02:13.25 than the strategies used in number 1, number 2 and number 3.
00:02:17.11 So, let's go ahead. It's now been more than twenty years since we had the first field trial
00:02:23.16 of a genetically engineered, disease resistant food crop.
00:02:28.00 My lab was involved in the collaboration with the Monsanto company
00:02:31.20 because very early on, we didn't have all the technology necessary to develop transgenic plants
00:02:36.22 in our own labs and we collaborated with scientists at Monsanto to develop the plants that you see here.
00:02:43.18 These are tomato plants, they carry a capsid of protein of a simple virus, tobacco mosaic virus.
00:02:50.16 Tobacco mosaic virus and tomato mosaic virus are cousins.
00:02:53.20 And these are, this is the result of infection of these ten plants on this slide with the virus in our greenhouses
00:03:03.06 and you can see that the plants here are growing much taller and are much more healthy looking than the ones below.
00:03:09.25 These are resistant to virus infection and what we found in 1985 was that we could reproduce the work from tobacco
00:03:17.11 to tomato to petunias and other plants.
00:03:21.02 We then were able to, through applications made to the US Department of Agriculture
00:03:25.26 and the Environmental Protection Agency, to plant these in the field.
00:03:28.23 And it's been now more than twenty years, my hair is not brown anymore,
00:03:34.18 it's more light colored, but I've grayed because of a lot of things.
00:03:40.29 And one of those challenges that I find still curious is how technology like this one and others
00:03:47.11 have still found it difficult to be released into the commercialization stream.
00:03:55.27 To date, we have only one success from an academic laboratory
00:04:00.08 of virus resistant plants that have been developed through these kind of technologies.
00:04:05.15 That's a papaya that was developed at the University of Hawai'i.
00:04:10.01 And there's now a squash variety that's also on the market from the public sector, from the private sector.
00:04:16.25 So, why hasn't there been more success?
00:04:18.19 Is it because we don't know enough about the virus?,
00:04:21.22 or is it that we don't know enough about how to do regulations?,
00:04:25.11 is it what we don't know about commercialization,
00:04:28.12 why has the public sector not been more involved in this science and technology?
00:04:33.01 Because as you'll see, what we can contribute is significant.
00:04:36.13 And we need to learn how to use our knowledge to enhance the contribution of our science
00:04:44.16 to food production in this world where population will continue to grow,
00:04:50.09 where ever more pressures of food production will continue to grow as population grows,
00:04:55.25 where there's still only a certain amount of land that can be planted,
00:05:00.02 only a certain amount of fresh water that can go onto agriculture lands, and yet population continues to grow.
00:05:05.22 The challenges of plant sciences include those, the studies of basic mechanisms of disease,
00:05:12.00 and finding ways to interdict those diseases, either through classical genetics and plant breeding
00:05:17.09 or through, perhaps, through biotechnology and what I want to talk about today are specific examples
00:05:22.14 where biotechnology can be used, or has been used, to develop plants that have resistance to the pathogen.
00:05:29.13 Our next challenge is to get these tested under confined field trial situations
00:05:35.29 to evaluate them not only in laboratories, in greenhouses, but in actual field situations,
00:05:41.11 as has been done in this first case, now more than twenty years ago.
00:05:44.21 What I hope is that there will be more and more examples of the public sector, such as we are,
00:05:50.13 to developing products that will be useful in release for people to increase their food production.
00:05:58.14 So, first a little bit of review of what has been done in scientific laboratories to develop resistance to viruses.
00:06:08.07 There's been a variety of strategies. Some of these are called pathogen derived.
00:06:12.06 That is, we use genes and open reading frames or small RNAs
00:06:16.26 to develop strategies to stop the virus in its process of virus replication.
00:06:22.24 And there are a variety of those,
00:06:24.06 in some cases, it's possible to use the genes that come from the coat protein sequences,
00:06:29.10 as we did for the tomato mosaic virus case.
00:06:31.21 Or, using replicase-related proteins, those enzymes that are required for replicating the viral genome.
00:06:39.13 In other cases, their strategies have been to block the function of the movement protein,
00:06:43.11 the latter has not been very successful to date, but I hope through the last lecture,
00:06:48.11 you gained a sense of how much we know about movement proteins,
00:06:52.02 and the more we know, the possibility of blocking their action, perhaps having a wider array of effects on virus infection.
00:07:00.28 The complementary strategies are to use sequences from the viral genome that will induce the formation of small RNAs
00:07:08.07 that can then silence, and lead to the degradation of viral RNAs during the replication process.
00:07:14.24 This seems to work quite well in some cases, but not in others.
00:07:17.24 Now, the challenge here is that these, it's imagined or expected that the RNA mediated resistance events
00:07:24.26 will work in those cases where there's very little divergence in viral sequence.
00:07:29.29 As you know, silencing complexes are formed because of the close sequence similarity between one RNA and the target RNA.
00:07:40.12 If those diverge because the divergence in viral sequences, it might be a challenge for this kind of approach.
00:07:47.25 Nevertheless, RNA mediated resistance can give very high levels of resistance in transgenic plants.
00:07:53.08 That will not be the topic of my talk today, we've been working primarily on protein mediated events.
00:07:59.23 There are other examples that are being used in which resistance is derived not from the pathogen sequences,
00:08:06.06 but from other sequences.
00:08:07.21 For example, I'm going to show you a case in which we've used transcription factors
00:08:11.22 to restrict the replication and disease caused by rice tungro disease.
00:08:16.15 In some cases, there are single-stranded DNA binding proteins.
00:08:20.03 Other strategies have re-created interferon like strategies in plants or using, developing plants that have monoclonal antibodies
00:08:29.00 that would bind specific viruses or virus proteins and slow down their virus replication by doing such.
00:08:35.17 There is a whole series of data that now indicate that some of the translation factors that are involved
00:08:43.17 in translating viral RNAs can be used to enhance disease resistance in certain cases, classes, of viruses.
00:08:50.27 So, again, the more we know about the virus and how it replicates,
00:08:54.18 and the more we know about the host and how it responds to virus invasion,
00:08:58.18 the more successful we'll be to develop strategies for resistance against infection.
00:09:03.09 So, I want to talk about a model system, the coat protein mediated resistance.
00:09:09.03 How does it work? Why does it work? And how can we make it more effective?
00:09:13.25 I want to give you a real-life example of a virus where we've used a coat protein to protect against tobacco streak virus in India,
00:09:22.03 it's a real world application of the technology that's now more than twenty years old.
00:09:25.21 Rice tungro disease, using novel approaches because the approaches up here didn't work in the one on rice tungro,
00:09:32.00 we found out about ten or twelve years ago.
00:09:33.29 And second, then lastly, I want to talk about a protein that we call G5 protein,
00:09:37.22 it's a protein from an inovirus, a bacteriophage, to control gemini virus replication.
00:09:43.25 Now, there are lots of plant viruses, lots of animal viruses, lots of viruses of phage,
00:09:49.25 and in this virosphere, developed through the International Committee on Virus Taxonomy,
00:09:55.16 and Claude Fauquet and his colleagues, developed this virosphere, I won't go through it,
00:10:00.05 but I want to talk about this group of viruses at this point. The virus I'm going to talk about is tobacco mosaic,
00:10:05.21 it belongs to this class of single stranded RNA viruses that are the subject, was the subject of my previous lecture.
00:10:15.13 A little primer on tobacco mosaic virus. As I indicated earlier, we know quite a bit about how it's replicated
00:10:21.25 from the molecular perspective. We've learned a lot about how the virus replicates in the cell,
00:10:27.14 where it works in the cell, how it forms these replication complexes and talked about how the virus might move from cell to cell.
00:10:34.05 Now, in 1985 we discovered that when we introduced one of the genes from the virus, the capsid protein gene,
00:10:41.28 into tomato plants, we had a population of plants that were resistant.
00:10:46.23 In fact, the level of resistance was correlated with the level of capsid protein.
00:10:50.22 The more capsid protein, the more resistant.
00:10:53.14 As we did our early studies, in the 80's and early 90's, our model that we developed, supported by extensive data,
00:11:01.18 indicated that the coat protein, if the coat protein is in large enough amounts in the plant
00:11:07.07 and if it could self-assemble to make these trimers that I also show here,
00:11:11.25 and these two, or this one, were responsible for binding to virus as the virus entered the cell.
00:11:20.04 And sort of re-capped the virus and not letting the viral RNA out of its shell.
00:11:24.29 If it can't get out its shell, of course, it can't bind to ribosomes, it can't start the infection process.
00:11:30.18 So, in this, in the first few years, as we came to realize that coat protein mediated resistance,
00:11:38.04 was working by blocking the disassembling of the virion and therefore stopping the virus in its tracks.
00:11:43.26 One of the ways that we showed that this might be the case is that when we inoculated these plants that were transgenic
00:11:49.05 with the, that had the coat protein, with the naked viral RNA without the capsid protein,
00:11:54.06 we could overcome the resistance. And I'll come back to that a little bit later,
00:11:57.26 because we used that strategy to study virus replication in cells as well.
00:12:02.08 So, that led us to believe that at least the earliest events in virus disease blocking was caused by a re-capping of the virion.
00:12:11.05 Because Gerald Stubbs and a large number of other laboratories have worked on the structural biology of the virus
00:12:17.07 we were at quite a positive advantage in our studies.
00:12:21.05 What we said is, if virus, if interaction of proteins with each other, the capsid proteins with each other,
00:12:27.02 is important for virus disease resistance, perhaps if we block the ability of these capsid molecules to assemble,
00:12:35.24 or we made mutations that would increase the affinity of the proteins, one for the other,
00:12:40.22 we would increase the resistance level.
00:12:42.19 And I want to show you the outcome of those studies.
00:12:46.05 Now, we used the residues in the structure in this ribbon diagram of two capsid molecules,
00:12:55.09 we know how they're interacting, you see here two acidic amino acids,
00:12:59.27 aspartic acid at position 77 and glutamic acid at position 50, on two different capsids.
00:13:06.00 These should be repulsive, but there's a calcium that lies in between and neutralizes those negative charges
00:13:14.15 and allows it to stay together. Actually, one of the points in the early stage of virus infection, the calcium is released,
00:13:20.26 allowing these to separate from each other and thereby releasing the viral RNA.
00:13:25.16 We're also fortunate to have the quaternary structure of the assembled virion,
00:13:30.01 so we know how they interact, the subunits interact with each other, side to side and top to bottom.
00:13:35.15 It's been really helpful as we've looked at sort of molecular structures and the effect of structure on resistance.
00:13:41.03 So, here are the results of a very simple experiment.
00:13:44.14 What we did was make a number of mutations in these specific amino acids of the capsid protein
00:13:51.22 and then put the gene for the capsid protein back in the virion and asked if the virion would assemble,
00:13:58.09 if it would, if the virus could move cell to cell, if it could go from leaf to leaf,
00:14:02.25 as I talked in the last lecture.
00:14:05.08 But we also put those mutated proteins into tobacco plants and made populations of transgenic plants
00:14:13.10 that had the virus protein.
00:14:15.09 We made something like fifteen independent plant lines for each of the mutations,
00:14:22.06 so you can see we made a lot of mutants,
00:14:24.10 and then identified out of that population those lines that accumulated the same amount of the protein,
00:14:31.07 the wild-type protein, or the mutant protein. And then used these in a plant pathology study,
00:14:36.22 simply challenged these lines with virus and watched them get sick.
00:14:40.28 So, these are non-transgenic lines, you can see that these plants all became diseased in a certain period of time.
00:14:48.22 And the more sick the plants they look, of course, the more virus they have in them.
00:14:53.05 So, these are two independent plant lines that have two, I mean, two examples of two lines that have two different mutations in them,
00:15:00.03 in the coat protein. And you see that there might be a little bit of delay between the onset of disease
00:15:06.19 in this line, compared to this line, but clearly they're not happy and they show disease.
00:15:13.20 But now, jump down to this other group.
00:15:17.14 Here's a group of transgenic lines that you can see have the mutant coat proteins,
00:15:25.01 this line 3646, is the historical line that we've used for very many years.
00:15:29.00 And you see here that these plants are different than the ones up above.
00:15:35.00 In fact, they show some disease, but the onset of disease is later, it's delayed,
00:15:40.19 and it's not as severe. Then there are another broad set of mutations in the coat protein
00:15:47.02 that either don't get sick or get sick very, very late in the infection process.
00:15:52.07 How did they manage to escape the infection? What's the nature of these mutations
00:15:55.26 and how might they delay the infection process?
00:15:59.03 Now, we've not done all the experiments that I'll tell you about with all of them,
00:16:03.00 we've focused on T42W, one of the lines that we've studied the most, but it's emblematic of the others
00:16:10.00 and we have other data that indicated this group really acts as a group and I'll talk about those studies.
00:16:15.12 In this process, we also have made transgenic cell lines in the cell line BY-2, it's a tobacco cell line.
00:16:24.01 Now, the importance of those cell lines, then, is that we could make single cells, or protoplasts, from them
00:16:29.13 and study single cell dynamics of virus infection.
00:16:33.13 And you see that here, in this case, we did the infection of the cells
00:16:37.19 and you see that there's, they were inoculated at zero time, then we took samples at different times after infection
00:16:45.11 and looked for the accumulation of virus proteins.
00:16:48.00 So, you see that in this case, these sets of mutants, there was no, there was replication and accumulation
00:16:55.08 of the coat protein, just as in the non-transgenic cell line.
00:16:59.28 Here's another set of cell lines in which there was no infection or very low level of infection,
00:17:06.06 as shown by the accumulation of coat protein.
00:17:09.22 Basically, this subset of cell line lines, which showed less accumulation of coat protein
00:17:16.10 also showed less accumulation of movement protein or of replicase.
00:17:20.00 They can be divided into two categories; those where there was high susceptibility, high susceptibility,
00:17:25.29 and less susceptibility, or greater resistance.
00:17:29.01 Why was that? How did that work?
00:17:30.16 And why does the coat protein function differently in different cases?
00:17:35.02 So, then we went back to the cell biology of how the virus infection and replication complexes is set up.
00:17:41.23 As I indicated in the previous lecture, the red color represents where the virus replicase is located,
00:17:49.00 the green, where the movement protein is and the blue where the coat protein is localized.
00:17:55.18 This is relatively early in the infection cycle, you can see that these green bodies representing some of the virus replication factories
00:18:02.23 are relatively modest in size and have not coalesced, as at later stages.
00:18:07.20 But, if this is what happens, we then looked at these different cell lines that we had produced
00:18:13.28 and we went through the whole process, we can chart the accumulation of the movement protein
00:18:20.23 as an early stage of infection, a mid-stage of infection and later stages of infection.
00:18:26.04 You notice that here, the puncta are very small and that represents the accumulation of the movement protein
00:18:33.07 using a movement protein fusion with green fluorescent protein.
00:18:36.19 In this case, we've challenged each of these cell lines with virus RNA,
00:18:42.06 so we could overcome the resistance reaction from the coat protein and now we're just now following infection.
00:18:47.24 So, you can see that these bodies grow larger in size,
00:18:51.02 they really get larger, and then down below, in the later stages, bodies look quite large,
00:18:56.02 and then, through a proteolysis of the movement protein, targeting degradation,
00:19:01.08 and the bodies are not visible anymore through the green fluorescent protein.
00:19:06.11 They can only be seen by electron microscopy and localization of the replicase.
00:19:10.16 So, we then began to categorize these cell lines in certain ways
00:19:15.06 and say, do they all progress from early, mid to late stages at the same rate?
00:19:21.27 So, a very talented research assistant looked at these cell lines at different hours after infection,
00:19:28.12 at ten hours after inoculation, at different time points,
00:19:32.26 and then decided if they were in early stages, mid stages or late stages of virus infection.
00:19:39.07 And what you see here is that there are a set of cell lines that all act as if they are non-transgenic.
00:19:47.01 They go through progression of disease and accumulation of these bodies
00:19:52.07 just as a non-transgenic cell line would do.
00:19:54.27 There's another set of mutants that are demonstrated here that look like they progress through the infection cycle
00:20:02.23 in much the same way as does a cell line that makes wild-type coat protein.
00:20:07.15 This is the first time we had noted that this process is different in this cell line than in this cell line up here.
00:20:14.08 Notice how it goes through that progression very rapidly.
00:20:18.05 What that indicated to us is if you get virus infection in these cells
00:20:21.25 by avoiding the disencapsidation, or the release of viral RNA, the infection process can even go faster than normal, than expected.
00:20:29.20 That indicated something very special and that is that the capsid protein was acting, perhaps,
00:20:34.16 as a positive regulator during the replication cycle. That's something we hadn't discovered before.
00:20:39.18 And others of these protein mutants act also as positive regulators,
00:20:45.21 but then down below you see another whole group of cell lines.
00:20:50.23 In those cell lines, they mostly get trapped in the early stages of infection, this stage,
00:20:58.02 or the one just down below.
00:21:00.03 Why was that?
00:21:01.14 That indicated that these, to us, or suggested to us, that these mutant coat proteins
00:21:07.14 were actually acting as negative regulators, not positive regulators.
00:21:11.01 And so we went back to our cell biology.
00:21:13.06 Now look at these two slides and what you're seeing on the left hand slide is an infected cell
00:21:20.05 that has one of these mutants that act as a negative regulator.
00:21:23.07 Notice you find almost no, there's almost no movement protein there.
00:21:27.02 It's making mostly capsid protein and viral replicase, a little bit of movement protein, but not a lot.
00:21:35.04 And only on the other side of this slide is the non-transgenic cell line, which you see very, very large replication bodies,
00:21:43.05 just as I showed you in the previous lecture. These bodies are able to move around rapidly within the cell,
00:21:49.24 they are able to move cell to cell in an infected leaf.
00:21:52.11 So, here was the first indication that this is a negative regulator in some way,
00:21:57.13 and blocking the production of movement protein. And here's the clue:
00:22:02.13 if there's less movement protein, there's less ability of the virus to move from cell to cell.
00:22:07.08 So, you can see that you can have blocking of a process that results in disease resistance.
00:22:13.10 So, what's the structural biology of that whole, what makes the structure of that high-resistance conferring mutant
00:22:22.18 different from the structure of the capsid protein, and how might that structure affect function.
00:22:28.22 I told you earlier, I showed you earlier, that the virus is really a helical array of about 2,140 of these capsid protein molecules.
00:22:39.00 They all stack together and wrap around the viral RNA to produce the virion
00:22:44.00 and they do so so it looks like a helical stack.
00:22:46.24 Here's a, this stack, however, makes a view in the electron microscope that looks a lot like this.
00:22:55.02 These are cryo-EM reconstructions, or pictures, of virions.
00:22:59.22 Notice that this part here is notably different than this. And in fact, if you look at the Fourier transform
00:23:07.05 of the incidence of reflection after, you find that this one is different than this one, they're spaced differently.
00:23:15.17 That spacing was, that information was then used by Tom Smith at the Danforth Center
00:23:20.28 to help us model this structure and what Tom concluded in his modeling is that this structure is different than this one.
00:23:32.10 Essentially, the top, if you represent this as the top of the protein and the bottom of the protein,
00:23:36.17 the top and bottom, you have an A/B, A/B arrangement up here.
00:23:41.07 But what's happened here is that you're having an A/B, B/A arrangement.
00:23:45.29 And the suggestion is that this may be a negative regulator, whereas this is not a negative regulator.
00:23:52.23 Interestingly, Gerald Stubbs, nearly 20 years ago, in his electron microscopic view of some of these virions,
00:24:01.16 in the way he did his science, suggested that there might, some of these occurred in natural virus infection.
00:24:08.14 So, I think we suggest that what's happened is that the mutations that we developed
00:24:13.22 create a, change the incidence of this and this, and the more of this, the more resistance there is.
00:24:25.20 We know that these globs, these assemblies, these platforms, can interact with these.
00:24:32.29 You see it in this structure here, some of these are A/B, A/B and some are A/B, B/A, co-assembled.
00:24:42.25 That was an interesting, that model was very interesting and important, if you were a structural biologist
00:24:49.17 these two images in C and in G can be viewed as stereo pictures. So, here's the model.
00:24:58.06 During virus infection, the replicase binds to the, first part of the infection process produces the enzyme, the replicase.
00:25:08.01 That replicase then structure, binds to this end of the viral RNA and makes the complementary RNA.
00:25:16.16 It then goes to points on that complementary RNA where there are appropriate structures,
00:25:23.04 they're internal promoters, they're shown here as stem loops,
00:25:27.14 and the consequence is that this enzyme then makes these smaller RNA molecules that act as messenger RNAs
00:25:34.27 to produce the movement protein.
00:25:37.14 Other such genomic RNAs make more capsid protein. In the presence of the wild-type capsid protein,
00:25:44.27 you have an enhancement of this promoter region and make lots more movement protein and coat protein messenger RNAs.
00:25:52.20 However, we think that, we propose that when the mutant coat protein and the wild-type protein bind together,
00:26:00.12 they block that receptor, or that promoter, or the binding of the replicase protein to that structure.
00:26:08.16 In any event, you get much less movement protein being made. Some made, but not very much.
00:26:12.27 As a consequence, you don't have as much movement protein. As I talked about in the last lecture,
00:26:17.24 those movement proteins are absolutely essential for moving these virus replication factories around the cell.
00:26:24.22 Here's a virus replication factory that's produced, but it doesn't have movement protein.
00:26:28.09 It also doesn't move over here unless there's a little bit of movement protein stuck into it.
00:26:33.23 So, these factories move around because of the availability of the movement protein in right place, at the right time,
00:26:41.00 to bind to these, to the transport mechanisms, over to plasmodesmata.
00:26:46.15 So, that's the model of how resistance might work.
00:26:49.11 So, over the last fifteen, twenty years, we've learned a lot about coat protein mediated resistance,
00:26:55.08 as these fly-ins show. As you read through them, I think, I hope I've covered and given some,
00:27:02.16 covered each of these topics and hope you can understand where we are in our understanding of how the coat protein can act as a positive regulator,
00:27:09.28 or as a negative regulator. Now, those of us who are interested in protein structure and function
00:27:14.18 can take heart from this, because there are some virus proteins, some virus capsid proteins
00:27:20.08 are only positive regulators.
00:27:22.03 The more we know about those positive regulators, it'd hold, that we could learn more about how to make a negative regulator out of that positive
00:27:29.05 and use that knowledge to strategize, to develop approaches to develop disease resistance,
00:27:34.28 so that we can have more coat-protein mediated resistance that's more durable.
00:27:40.01 I'm going to show you a brief example of an application of this technology, now twenty years old,
00:27:46.11 this is a collaboration between my laboratory and a laboratory in Hyderabad, India,
00:27:51.23 this is ICRISAT, it studies the crops that are important in southern, tropical areas of the world
00:27:59.28 and in south India, of course, is quite tropical, very arid, and that's their focus,
00:28:06.05 as well as focusing in Africa. Here's an example of a disease in groundnuts that is really very devastating,
00:28:13.03 it can be very devastating and it's transmitted by an insect called, I mean an arthropod, thrips.
00:28:20.02 And it carries the pollen from one plant to the next, and the pollen, on the outside of the pollen,
00:28:25.10 is the virus and as the insect chews on the leaves, it transmits the virus from the pollen's surface into the leaf structure,
00:28:32.29 into the cells and off goes the disease.
00:28:35.16 But it's very devastating and the impact can be quite remarkable, as indicated by some of these headlines in the newspaper.
00:28:43.18 The disease was first identified in the mid-90's, it recurs depending upon weather and humidity,
00:28:54.16 and rainfall and you can see that it affects the area of India that's sort of the middle, downward,
00:29:00.09 the state of Maharashtra is up here, Mumbai, and then on down through Tamilnadu.
00:29:05.29 And what was important in our realization was that this disease not only affects groundnuts, or peanuts,
00:29:12.29 but it also affects other crops. And we found if we could have a strategy that helped develop resistance against this virus in peanuts,
00:29:20.06 we could also apply that in other crops or other institutions can do so.
00:29:24.08 So, this is an example, the virus affects sunflower and if you know Indian agriculture,
00:29:31.11 you know that sunflower and groundnuts are main sources of vegetable oils that are used in the cooking in India.
00:29:38.04 These two diseases have dramatically reduced the amount of cooking oil, and they have to import cooking oil
00:29:43.25 from other sources, including palm oil and soy oil.
00:29:47.20 The disease also affects melons and cotton, and marigolds, it affects the floriculture industry as well.
00:29:57.25 What was really interesting to us is that these viruses that are in these different groups of plants
00:30:03.08 are very similar to each other in sequence. We're very optimistic, then, that if we could solve the disease in peanuts,
00:30:09.22 we could maybe have some impact on these other diseases.
00:30:13.26 This virus is very different from tobacco mosaic virus, but there was a history of study of this virus
00:30:20.22 soon after we described coat protein mediated resistance against the tobamoviruses,
00:30:27.11 TMV and tomato mosaic, colleagues in Europe asked the question of whether or not the coat protein resistance
00:30:35.29 would work against these classes of virus, this is a group of viruses called Ilar, a group to which other important diseases belong.
00:30:45.07 The genome structure of this virus is quite different than TMV,
00:30:48.10 there are three different RNA molecules packed into this circular particle
00:30:53.04 and the coat protein is actually made as a sub-genomic RNA that comes off of RNA3.
00:30:58.14 We worked to develop a gene construct which was then made available to our colleagues in India.
00:31:06.27 I should say that before we did the work, we had free, full freedom to operate with all inventiveness
00:31:13.21 and we had agreements with those who help the relevant, important patents on the processes and the technology, per se,
00:31:21.12 that if we were successful, these products would be released, free of charge,
00:31:25.10 without any royalties or license fees to the farmers in India who needed them.
00:31:30.02 Kind of a philosophy that we in the academic sector can apply under a variety of conditions.
00:31:38.18 Here the slides show that the effect of infecting transgenic peanut plants with this virus in greenhouse conditions.
00:31:47.29 Our colleagues in India have developed a large number of transgenic lines
00:31:52.01 and now are evaluating them for the genetic structure and for the resistance.
00:31:56.03 They have a number of lines. This is a line that has a delay of infection, but not a high level of disease resistance.
00:32:05.25 These lines, on the other hand, are highly resistant to infection,
00:32:10.21 these cell lines, these plant lines also have higher level of coat protein than those that are less resistant.
00:32:16.08 So, it's a coat protein mediated resistance phenomenon, we're hoping that we'll be able to extent this work
00:32:23.17 into more greenhouse studies and eventually into field trials in the next year or two.
00:32:29.19 So, I'm pleased that the technology that's now more than twenty years old can actually be used in crop situations,
00:32:35.04 in India and we think in other parts of the world as well.
00:32:38.24 So, how does it work for this virus? Is it like TMV?
00:32:41.29 That we don't know, it's not been studied to the same detail at all, in the case of the resistant lines.
00:32:49.01 But some work that was done by Lee Gehrke's lab and published earlier in Science,
00:32:53.23 provided a structural model for how the capsid protein of this virus binds to,
00:32:59.16 or a virus that's like tobacco streak/alfalfa mosaic virus, ALNV, is an alfalfa virus,
00:33:05.28 and with a similar strategy for replication. And Lee and his colleagues have looked at the structure
00:33:11.22 of the complex of coat protein with the viral RNA and found that, yes indeed,
00:33:17.07 as we have known from the biological experiments that were done over the last twenty years,
00:33:21.18 that in fact, the capsid protein binds to the 3' end of the viral RNA
00:33:26.05 to some of these hairpin loop structures and important for regulating replication.
00:33:30.07 The challenge is that we know the coat protein is a positive regulator, it's essential for virus replication.
00:33:36.26 So, how does it work as a negative regulator? That we don't know.
00:33:40.18 Whether it creates specific structures, or intermediates that block its function as a positive regulator
00:33:47.04 we simply don't know and we need some ongoing work to figure out how this works.
00:33:51.16 I want to talk about a very different virus.
00:33:54.16 This is a virus that's a pararetrovirus. The disease is actually caused by co-infection of two viruses,
00:34:01.05 one that's spherical, contains RNA, one that's bacilliform.
00:34:04.20 These two together can be transmitted by the insect vector, a green leaf hopper,
00:34:10.03 and if transmitted to a young rice plant, that's shown in this case,
00:34:14.10 that the result is that it causes a dwarfing of the plant, it stunts its growth,
00:34:21.02 and accumulates large amounts of virus particles in the phloem tissue and causes this severe discoloration.
00:34:28.13 In severe cases, the infection is so strong that it prevents development to the point there is no panicle made
00:34:35.17 and if no panicle, there are no rice seeds.
00:34:37.25 I've seen fields in India where the disease is in the range of only, it reduces the output, the yield of the crop
00:34:46.29 by as much as 80 percent. It's periodic, it depends on the availability of the insect
00:34:54.16 and whether or not there are infected plants in the region.
00:34:57.18 This virus is responsible for making the proteins that allow the insect vector to acquire this virus,
00:35:07.04 as well as this virus. Now, for a number of years, we've studied coat protein mediated resistance against this virus,
00:35:13.22 and this one's an interesting virus because it contains three capsid molecules,
00:35:18.04 it looks more like some of the animal viruses than any other plant virus that we know about.
00:35:23.10 But the level of resistance provided by coat protein, or capsid proteins, in transgenic rice lines, was low.
00:35:30.11 It did reduce the amount of replication and disease, but in fact, we came to know that this virus is the responsible agent for the stunting.
00:35:39.09 We then began to attack this, to learn more about how this virus works.
00:35:43.15 And through some work of a number of outstanding colleagues, we learned, we've been tearing this virus apart
00:35:53.17 and learning how the promoter works and how replication goes on.
00:35:57.06 This is a virus that doesn't live in the US and so, all experiments related to it need to be confined to controlled environments
00:36:06.24 and locked chambers and so forth. It belongs in a class of viruses called pararetroviruses.
00:36:11.29 And the life cycle of a pararetrovirus is very different than the life cycle of tobacco mosaic virus.
00:36:17.14 The virus enters the cell, and by, again, in this case, by feeding from the insect,
00:36:21.19 and then you can see that after disassembly, the genetic information, the double-stranded DNA,
00:36:29.07 goes into the cell nucleus. Unlike other pararetroviruses, this one, there's no evidence that this one integrates into the viral genome.
00:36:36.04 But here it forms a minichromosome and that results in the product of more double-stranded DNA,
00:36:43.20 which is transported by an unknown mechanism, goes into the cytoplasm
00:36:47.13 where gene expression leads to the production of capsid proteins and movement proteins and so forth
00:36:51.19 and then the assembly of more virus particles.
00:36:54.22 So, it's very different than tobacco mosaic virus or other RNA-containing viruses.
00:36:59.14 The genome of the virus is a circular double-stranded DNA virus.
00:37:05.10 The promoter region, which is responsible for making this full-length transcript, shown in this dark band here,
00:37:11.15 is in this region. Now, in studying that in rice plants and in other plants,
00:37:17.15 we found, we came to know that the promoter that is in this region,
00:37:20.26 when linked to a reporter gene, shows expression of that promoter only in vascular tissue.
00:37:25.29 That's in line with the observation made by plant pathologists that the virus accumulates in vascular tissue.
00:37:32.20 In our studies and subsequently of the promoter region, we came to identify a unique region in this promoter sequence
00:37:40.28 that was unlike other sequences that had been described as promoter related sequences.
00:37:47.13 And we called it Box II, it's proximal to the TATA binding protein site, binding site.
00:37:55.15 Through the standard single hybrid, and two hybrid kinds of procedures, we identified proteins that bound to that promoter sequence.
00:38:06.04 These we call RF2a, rice factor two a, rice factor two b.
00:38:12.07 These can form binding, these can bind to that Box II sequence I talked to you about earlier,
00:38:17.12 either as homodimers or as heterodimers. Both of these are activators of transcription.
00:38:22.20 We characterized the function of each of these domains, proline rich, glutamic acid rich and this glutamine rich domain,
00:38:30.28 and the acidic domain and so forth.
00:38:32.26 Down here is an additional protein, this is RF, this is a protein that we call RLP1.
00:38:40.02 It also is a bZIP protein, but it does not bind to the DNA binding site alone;
00:38:46.15 it can only bind in the presence of either RFIIa or RFIIb.
00:38:50.10 But it's a negative regulator.
00:38:52.26 So, our model is that, as you know, these bZIP domains recognize a unique binding sequence
00:39:00.09 and as that sequence is bound and does its function,
00:39:03.13 and one the functions, apparently is that, in binding, it can interact with TATA binding protein,
00:39:08.18 we showed that there is some specific interaction between the glutamic acid domain of these proteins
00:39:14.07 and the TATA binding protein that presumably plays some role in bringing the holoenzyme to bind on that promoter.
00:39:21.03 Once it binds, it makes the messenger RNA and off goes the translation and then process of replication and packaging that are necessary.
00:39:30.13 But the characterization of these transcription factors was an interesting part of the exercise,
00:39:36.09 but we then had some ideas about how we might use that information.
00:39:40.03 So, here's a hypothesis: if the replication strategy involves these transcription factors,
00:39:49.10 how do these, what are the roles of these transcription factors in plant development?
00:39:53.09 So, we placed the experimental emphasis on the study of the promoter
00:39:58.23 and we, and overexpression of the transcription factors in transgenic lines.
00:40:03.09 For example, here's a transgenic line that has, in which we've reduced the amount of the one transcription factor
00:40:11.06 by using an antisense gene construct. So, a transgenic line that doesn't look very happy, it's stunted.
00:40:16.01 It doesn't have many roots either.
00:40:18.10 But it can outgrow it with time. And eventually, it will produce a small amount of seeds.
00:40:24.28 In contrast, this is another in which we've overexpressed RFIIb, the second transcriptional activator.
00:40:34.09 And you see that those plants can get through this early process, but they don't continue further.
00:40:40.10 They get stunted. In fact, some of the coloration of the leaves look interesting,
00:40:44.02 in that they lack some of the pigments and they have a little bit of red tinge on them.
00:40:50.04 This is what happens when we overexpress RLP1, the negative regulator.
00:40:55.11 When we knock that gene down, that amount of RLP1 down with an antisense gene construct,
00:41:01.13 we saw no phenotype. We did see a phenotype when we overexpressed the amount of RLP1.
00:41:07.21 Now, if we compare and contrast the symptoms of disease caused by the virus,
00:41:15.19 and any of these phenotypes of the transgenic lines, it looks most like this one, stunted and a little discoloration.
00:41:21.29 And we began to have the hypothesis that maybe virus infection was having an effect on development
00:41:28.01 by reallocating, or repositioning or reusing those transcription factors for replication of the virus
00:41:34.17 and not allowing the host to do its own development.
00:41:37.25 And this was further supported by this experiment. Shunhong Dai and his colleagues developed transgenic lines
00:41:48.06 that overexpressed the DNA binding protein of one of those transcription activators, RF2a.
00:41:54.18 Now those of you who know gene regulation know that when you overexpress the DNA binding domain,
00:42:02.05 it acts as a dominant negative repressor and it binds to the sites in the chromosomal DNA of the organism,
00:42:09.07 binds those sites to which the transcription factor would normally bind, and thereby not allow the transcription factor to bind there.
00:42:16.23 Now, when we expressed that dominant negative protein under the control of a promoter that would work in all cell types,
00:42:25.14 the ubiquitin promoter, we got a very strong phenotype of stunting.
00:42:30.07 When we use a different promoter, this one from the virus, rice tungro virus,
00:42:36.11 and overexpress this guy, we got also some stunting, not quite as severe, as you would imagine if you think about it.
00:42:44.06 It's not quite as severe. If we overexpress the dominant negative protein using promoters that are known to be expressed in the vascular tissue,
00:42:55.01 this is the shrunken 1 promoter from maize that we've used in this case,
00:42:59.07 and this is from a gene called phenylalanine ammonia lyase, also both of these are vascular tissue,
00:43:05.14 we got stunting. On the other hand, if we expressed that dominant negative protein in cells which are green,
00:43:13.06 these are the ones that are not vascular tissue by and large, they are the mesofiller tissue in the leaves,
00:43:18.09 finally, the plants are not stunted, and that, again, was a validation that the function of this, of proteins that have this domain,
00:43:30.19 are important for plant growth and development through growth and influence on the vascular tissue.
00:43:36.21 Remember that the virus is infecting the vascular tissue as well.
00:43:42.07 So, we then took these cell lines and asked whether or not they were resistant to virus infection or not.
00:43:49.10 And I'm going to show you two sets of studies. I want to show you next the result of a study
00:43:55.06 that was done in the Philippines by some colleagues and PhilRice, an institute that's funded by the government.
00:44:01.18 And they had the capability of feeding these plants on insects, with insects, that have been previously fed on virus infected plants.
00:44:10.29 And then we just looked at whether or not the plants got sick or not,
00:44:15.14 whether they were stunted.
00:44:16.18 And Dr. Alfonso and his colleagues showed that the plant was stunted, as expected,
00:44:24.01 and it's very similar to a susceptible variety that they use in the Philippines.
00:44:29.03 Now, the plant line for this pot labeled number 3 is a line of rice, a cultivar of rice, that's infected, resistant to infection, or eating,
00:44:41.20 by the insect. It's not necessarily resistant to the virus per se, but the insect doesn't like to feed on it,
00:44:48.01 so it's got a resistant capability because it's not fed on by the insect.
00:44:53.29 And then beside it, lines pot 4 and 5 are those plant lines that we have overexpression of the factor RF2a.
00:45:02.02 And those that have overexpression of the factor RF2b, and they're very much more like this line.
00:45:07.06 So, our plants didn't get sicker faster, rather they had some disease resistance.
00:45:14.16 When we thought about these experiments, talked about them in the lab,
00:45:18.27 we thought, well, maybe the virus would just use more of these replication factors,
00:45:22.21 or these transcription factors and replicate faster. It seems not to be the case.
00:45:26.27 So, we needed to show that, and with additional studies.
00:45:30.02 We then went on and learned how to do the virus inoculation, not with insects,
00:45:37.03 but in the laboratory with a special tool that we had, using agrobacterium to mediate the infection process.
00:45:44.08 And of course keep these in locked chambers.
00:45:47.24 And this is the result of those experiments that we had in our laboratory
00:45:53.13 and I want to show you them in contrast to what happened in the Philippines.
00:45:57.12 So, in this line, so, this represents the population of thirty plants that were inoculated.
00:46:04.17 In the case of plants that are control line, you see that after inoculation, down at the bottom corner,
00:46:13.04 and plotting this, by about 35 or 40 days or so, the plants cease growth.
00:46:20.12 They're stunted, they stop growing, just as we would have expected them to do.
00:46:24.20 The lines up above are a series of controls. This red line is a non-inoculated partner, this is the same line as this one,
00:46:33.10 but not infected, you can see how the plants continued to grow.
00:46:36.26 And then we show the RF2b and RF2a plant lines, and you see that the plant lines that are in the blue,
00:46:44.16 these are the two lines that are having the, that have the overexpression of RF2a, the transcription factor.
00:46:52.22 And they grow as well as, if not slightly better, than the controls that were not inoculated.
00:46:57.05 Likewise, the population of RF2b plant lines is indicated in this panel.
00:47:04.01 In this panel, we're looking at disease incidence in populations of plants.
00:47:09.25 This was developed by our colleagues in the Philippines. And in this case, you have a mathematical formula for disease incidence
00:47:16.22 and you can see that there's less disease incidence over a two month period by this plant line,
00:47:22.04 which has resistance to the insect. One of our plant lines, two of our plant lines are very similar in its resistance
00:47:27.22 to this model line that they're using as their internal control.
00:47:31.29 Here you see a bubble, indicating that these plants that are highly resistant do show a burst of disease symptoms in young leaves,
00:47:41.09 but then as the plants grow, they tend to outgrow it and the disease severity index is lower.
00:47:46.18 Other plant lines that contain the other transcription factor are a little bit different than these, but much less than the control.
00:47:53.14 Now, one of the possibilities that we entertained was that if we would make more of the transcription factor,
00:47:58.29 the virus would use it and you'd replicate more. And you'd get more severe virus, disease, than in the control.
00:48:07.20 And that clearly has not happened here.
00:48:09.18 But we didn't know about, anything about the molecular processes so Shunhong (Dai) and his assistants
00:48:17.22 worked to create cell lines from these transgenic lines and non-transgenic lines.
00:48:22.24 These cell lines could then be inoculated with the virus and we could study single cycle growth curves.
00:48:29.23 Now, I'll remind you that these are non-differentiated cells, they don't have phloem, they don't have vascular tissue
00:48:35.21 but they are de-differentiated for production of these transcription factors.
00:48:41.21 In other words, we expect that the transcription factors are made in these cell lines.
00:48:45.29 And then, of course, the transgene that we've introduced in these cases would introduce the over-production of RF2a,
00:48:55.08 as well as RF2b. What we show here in these panels are the accumulation of viral RNA in non-transgenic cells
00:49:03.06 and in transgenic cells.
00:49:05.25 It's clear that this band is lighter, there's less viral RNA in this, in these two lines then there is in the control.
00:49:13.10 That was a surprise. We had not anticipated that that would be the case.
00:49:17.25 Shunhong went on with the studies and this is really a place to say if you're a graduate student or a postdoc
00:49:25.03 and you have a great idea, go ahead and do it if you can.
00:49:27.28 I advised him not to do the study and he went ahead and really to my great surprise,
00:49:34.22 has revealed more detail about the resistance than what I had anticipated.
00:49:41.15 So, this is now, back using plants, not cell lines, but plants.
00:49:46.09 And these are plant lines that have been selected
00:49:49.14 and in this group there are thirty individual plants that are, the leaves are taken from thirty plants
00:49:54.20 and combined together, so if you count across here, you can imagine how many plants were grown in this experiment.
00:50:01.15 And you can see in the dark blue line that in the non-transgenic cells, or plants,
00:50:08.21 the amount of virus RNA accumulates to a high level at about twenty to twenty five days after inoculation
00:50:15.19 and then drops off and stays high. The transgenic cell lines that overexpress, overproduce the amount of RF2a and RF2b
00:50:23.01 have considerably less viral RNA than the controls and that this group has far less than this.
00:50:29.17 Well, how about virus itself? So, he did the experiment to do straight PCR instead of real-time RT PCR up here.
00:50:37.04 He then did the accumulation of viral RNA and true to fact, it's this line, this one set of lines that you see with the short bars
00:50:45.08 are highly resistant, not only to replication, not only to gene expression, but also to virus accumulation.
00:50:51.01 So, these lines not only look good, they have less virus in them.
00:50:55.11 Now, that might, you might anticipate then that these factors, these transcription factors
00:51:00.17 are not only affecting growth and development of the plant,
00:51:03.21 but they might have something to do with this transition from the susceptibility of a juvenile plant to the resistance of the more adult plant.
00:51:11.28 Now, perhaps by affecting the innate immunity pathway or other pathways that provide innate resistance to the plant
00:51:19.10 as it grows. The model that we have for the resistance reaction now is quite different than we had imagined
00:51:26.25 or that we saw in the case of tobamoviruses, tobacco mosaic virus. In this case, the insect feeds,
00:51:34.04 carries a virus to a young plant and as a virus invades, invades in the phloem tissue, as I talked about earlier,
00:51:43.01 where these transcription factors are, and we suggest that the transcription factors are re-allocated
00:51:47.14 to the replication and gene expression of the viral genome, therefore leading to stunting and disease
00:51:53.14 and that what we've done is shift that balance so that we now are able to outgrow the virus infection.
00:52:00.12 Now, the last group of viruses I want to talk about very briefly is very different
00:52:05.00 than either of the two examples I've shown before. This is a group of viruses that are single-stranded DNA viruses.
00:52:11.27 They're important in tropical, sub-tropical regions of the world, they cause severe disease.
00:52:19.10 They are characterized by a great deal of genetic variation
00:52:23.08 and a lot of sort of promiscuous crossing between the viral species.
00:52:30.03 They mix, they recombine, produce more pathotypes and disease can be more severe or less severe
00:52:35.26 depending upon the outcome of the genetic crossing, or mixing of the genetics.
00:52:42.00 This is a disease caused in cassava and this is a severely affected plant line, clearly it's very stunted.
00:52:50.17 The cassava plant produces large roots full of starch and is a sole source of sugar, or carbon,
00:52:58.20 for many hundreds of millions of people in Africa alone.
00:53:02.02 But normally, it would grow to a height of someplace above two meters,
00:53:06.21 if it's well fed, if there are no diseases. This one is a very short stem that's perhaps less than 20 centimeters.
00:53:17.16 The virus that invades, that's causing this disease, is a gemini. Gemini geminate because it has two particles,
00:53:24.21 one on top of the other and carry, this is a group of viruses that are called the old-world gemini viruses,
00:53:32.06 have two DNA components. And I won't go through the replication strategy of this virus,
00:53:37.00 it's quite different than those I've talked about earlier in this lecture.
00:53:40.13 One of the strategies that Dr. Fauquet and his colleagues have taken is using an siRNA strategy
00:53:47.02 based on specific sequences, determining what sequences are susceptible to siRNAs
00:53:52.12 and then engineering plants through genetic transformation to create plant lines
00:53:57.12 that can then be tested in greenhouses and growth chambers
00:54:00.11 and you that he was able to develop some virus resistant plants that have an siRNA strategy.
00:54:06.13 Now another strategy that he's taken is based on work that Dr. Bruce Alberts and his colleagues did
00:54:11.19 in the mid-1970's, when Bruce was in the biochemistry department at UCSF.
00:54:17.05 And in that case, we were studying an inovirus, a bacteriophage and identified a DNA binding protein, what's called G5,
00:54:26.10 and a post-doc in our lab a number of years ago showed that the G5 protein could be produced in E. coli
00:54:33.27 and could be shown to bind to gemini virus DNA.
00:54:36.21 He then did experiments to develop plant lines that would overexpress the G5 protein
00:54:43.02 and found, surprisingly, the ability to slow down the replication of gemini viruses.
00:54:48.26 That led, then, to additional studies in which we've improved the gene expression by variety of well-known tools
00:54:57.07 that are used by molecular biologists and then developed transgenic plants that carry and produce larger amounts of this G5 protein
00:55:06.04 and have some resistance. Now, the interesting part about this strategy is that the G5 protein does not bind based on sequence.
00:55:13.00 It's non-sequence specific. So, one would think that it would bind any single-strand DNA.
00:55:18.02 On one hand, you might think the protein would affect plant growth and development.
00:55:22.05 So far, we've seen none of that. But we have seen that the plant lines that contain this protein are resistant to a variety of gemini viruses.
00:55:31.25 For example, this is a cassava mosaic virus from Sri Lanka and that's quite removed from Africa
00:55:41.07 and the sequence of this virus is quite different from others in Africa.
00:55:45.08 Yet, the expression of that G5 protein gives resistance against this strain in comparison to the control.
00:55:53.28 It also works very well against a cassava mosaic disease strain from Uganda.
00:56:01.16 And one from Kenya, as shown by these slides.
00:56:07.03 Now, the, that gives us some hope that perhaps a protein like this would have pan-typic resistance against many kinds of gemini viruses
00:56:17.19 and that work is continuing and the current status of this project is that it's now moved from experimental phase
00:56:24.16 to one that we hope will lead to the development of material that can be tested in Africa by our colleagues in Uganda and Kenya
00:56:33.10 and other parts of the continent.
00:56:35.17 We have established strong partnerships in which their scientists come to the Danforth Center for research into gemini viruses
00:56:43.02 and developing biotechnology tools to detect the virus and then to develop lines that are resistant.
00:56:48.29 So, we're learning a little bit about what it means to be pre-commercial.
00:56:53.29 How do you do that? Academic institutions aren't very good at it, we've tried to learn from the private sector.
00:56:59.12 We have applications in place at the Institutional Biosafety Committees in Uganda and Kenya
00:57:08.27 with a cultivar that we call 60444. Now, the problem is that 60444 is a nice laboratory model
00:57:15.22 and it's used in some settings as a commercial variety for starch production,
00:57:20.18 but in fact it's not farmer preferred and if you've eaten some potatoes and yams and cassava,
00:57:28.21 you know that the varieties are different and you will like a certain variety, but not another.
00:57:32.18 So, the challenge now is to transfer the technology that apparently works in this model cultivar
00:57:37.13 into farmer preferred cultivars. So, our colleagues have identified those that are preferred most by the farmers
00:57:43.23 and we're now doing the construction and the work to transform that material, the best gene constructs, into those varieties that would be used by the farmers.
00:57:53.14 The project directors here are Claude Fauquet and Nigel Taylor.
00:57:56.25 Nigel is the specialist in transformation and regeneration of tissue and Claude the gemini virologist.
00:58:03.26 And we've worked now on this project, or they have worked on this project, for nearly twenty years,
00:58:07.18 with, now, some success. And with many colleagues that have come from Africa and Latin America to help.
00:58:14.08 But the job's not done and this is, I think, the penultimate slide.
00:58:19.15 This reminds us that the projects that happen in laboratories are a long way from the projects that end up in the field.
00:58:30.03 In the development of drugs that would have pharmaceutical application,
00:58:34.25 academics normally pass off their best leads to the private sector to help them develop them for commercial purposes.
00:58:42.19 And that's largely the case in agriculture biotechnology, we know the large companies have been very successful
00:58:48.25 in corn and soybeans and canola and cotton and so forth,
00:58:53.19 but the project that we're talking about here is a project that has little or no commercial value.
00:58:58.04 It's a crop that's important for those who live on 1 to 2 dollars a day.
00:59:03.27 They can buy enough for a dollar a day to feed themselves and their families, a family of four or five.
00:59:09.13 But that's all they might eat. And so this has little or no commercial value,
00:59:15.15 so we've done this in partnerships that have started in the US and will, we hope, end in fields in Africa or Asia.
00:59:25.14 But this is a long process. Our basic work, actually, started in 1988
00:59:31.14 and it wasn't until 1999 that we have the first transgenic line that looked promising.
00:59:36.10 But it failed under more stringent conditions.
00:59:40.18 Started all over again, re-engineered the plants, now those have been working for a number of years.
00:59:47.00 We're ready for our first, initial, confined field trials. These are field trials that are in a confined space
00:59:52.15 that no one has access to, that no material leaves.
00:59:55.19 It gets burned and trashed in that field.
00:59:58.09 If they're successful there, then it goes on to the next stage of trialing, in farmers' fields.
01:00:02.26 That can take more time. While you do this, in this re-iteration, you must work with the regulatory agencies.
01:00:09.12 Remember, the transgenic crops are regulated differently than plants that are developed through classical cross-breeding
01:00:16.00 or through mutation breeding, high energy neutron bombardment, and so forth,
01:00:20.29 breaking and rearranging chromosomes is something that's approved by the processes,
01:00:26.12 but genetic engineering at this point is not, it's regulated differently, so we as academics have to learn how to do this.
01:00:32.04 This is a very time consuming process and very expensive.
01:00:35.06 And that in part is what limits, I think, the involvement of the public sector,
01:00:40.18 such as the Danforth Center scientists or scientists, perhaps at your institution, from moving into real contributions in agriculture biotechnology.
01:00:48.26 But it takes time and it takes dedication and specific funding that will allow it to go on.
01:00:54.11 So, what have we learned and what have I told you today?
01:00:57.11 I've tried to describe a number of strategies that are being used to develop disease resistant crops.
01:01:03.27 We've made great process in virus resistance.
01:01:06.16 Other laboratories are making progress in developing resistance to fungal infections or nematodes, parasitic nematodes, and bacterium.
01:01:16.23 In our case, we've focused mostly on protein-based resistance reactions,
01:01:22.09 a lot of other laboratories are working on RNA mediated events.
01:01:25.05 Through this, we've learned a lot about what works and what doesn't work.
01:01:28.15 And as we've done so, we've learned more about the host, more about the virus
01:01:33.12 and hopefully have a greater chance of contributing to the production of food, and nutrition,
01:01:39.22 around the globe, in this, where plant sciences, basic sciences and the animal side can make contributions,
01:01:48.02 if we focus on the important of basic science and the translation to applications such as I've talked about today.
01:01:54.20 I hope you've found this interesting and if you want to contact me, please feel free to do so,
01:02:01.00 at the website that's listed on the first slide.
01:02:04.03 Thank you and I hope you have a good rest of the day.
01:02:06.11 Good luck.

Videos in this Talk

Part 1: Biology of Plant Virus Infection
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad
Part 2: Genetic Engineering for Virus Resistance in Plants: Different Viruses Demand Different Strategies
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Roger Beachy

Total Duration: 1:30:12

Recorded: April 2008

All Talks in Plant Biology

Talk Overview

This seminar describes the cell and molecular biology of plant virus infection. The first lecture will discuss how virus replication centers are set up in plants and how viruses use host cell mechanisms to facilitate cell to cell movement and eventual pathogenesis. The second part of the lecture will relate how this information can be used to produce crops resistant to specific viruses through different biotechnology strategies.

Speaker Bio

Roger Beachy

Roger Beachy is the founding president of the Donald Danforth Plant Science Center in St. Louis, Missouri where his research focuses on the cellular and molecular mechanisms of virus disease in plants. Before joining the Danforth Center, Beachy headed the Division of Plant Biology at The Scripps Research Institute in La Jolla from 1991-1998 and… Continue Reading