Good morning, thanks for joining the session this morning.
I'm Roger Beachy, a plant virologist and I work in biotechnology as well,
I'm from the Donald Danforth Plant Science Center in St. Louis, Missouri,
we're a new, relatively new, non-for-profit research institution,
and I want to talk to you today about viruses and plants.
I'm going to tell you a little bit about how they replicate, but more importantly,
the cell biology of how they set up the virus replication factories
and how the ability of the virus to use host mechanisms to move from cell to cell eventually ends up in pathogenesis.
The second part of my lecture, the second lecture,
will be related to how to use that information through biotechnology to develop virus-resistant crops.
So, I want to tie these two together, and hopefully what I introduce you today, in this first lecture,
will help you in the second.
So, what I want to talk about, again, is the cell and molecular biology of virus infection, replication and pathogenicity.
This slide shows several examples of virus infected plants.
The disease that's shown here results after the virus infects perhaps a single cell, or multiple cells,
spreads from cell to cell and then moves from one leaf to the next,
eventually invading the whole plant.
And as you see, the examples I have here are for cassava, in Africa, in papaya, throughout the world,
and this is an example of a disease on a tomato plant that shows severely affected plants in the front,
and disease resistant plants in the back.
Again, the result here is a culmination of events that leads from infection of a single cell
and spreading throughout the leaf and then throughout the plant.
Throughout my lecture, I'm going to talk primarily about tobacco mosaic virus.
It's a very simple plant virus, it's been used as a model for more than fifty years,
and scientists throughout the world have used it for a variety of studies.
I'm going to talk about it in terms of its cell biology and molecular biology of replication and spread.
There are lots of different examples of viruses in this category,
some in fact have hosts like tomato and peppers, but others have been identified that infect Arabidopsis and other model plants.
A little bit of a background.
The genome of tobacco mosaic virus is a single, very simple RNA molecule.
It encodes three genes. The first is an enzyme that helps the virus copy itself, it's an RNA dependent RNA polymerase.
The green box indicates the protein that's encoded by the virus to help it to move from cell to cell,
and the blue box indicates the location of the capsid protein.
The capsid, of course, its role is to replicate, is to encapsulate the single-stranded genome
and allow it to be transmitted from cell to cell, or from leaf to leaf,
and of course, that's what's transmitted as workers in the field propagate plants and move the virus around.
Now from the standpoint of molecular virology, this is a slide from Dr. Milton Zaitlin at Cornell University,
and you see that in the first iteration, a virus enters the cell, usually by  a wound,
either by an insect or a worker who might be working in the field,
transmitting the virus into a broken cell, and from there on, the molecular structures take over,
and processes take over, the virus is encoded, releasing the viral RNA, ribosomes translate it,
and those enzymes that are produced from the translation are responsible for copying the RNA
and building the virus replication factories to make more virions,
eventually ending up with more virus particles that are shown down there, down in the lower left hand corner.
Now, from a cell biologist's point of view, it's a very different story.
In this case, we've looked at how the virus enters a cell, and in the first iteration, the virion is dis-encapsidated
within a few seconds after it enters a cell.
Viral RNA is then moved into, on ribosomes, it's moved into the membranes that are perinuclear
and there it begins its translation and set up the first parts of the protein machinery that are necessary to produce more virions,
then moves, those membranes then move out into the cytosolic portion of the cell,
and set up larger factories, and continue to build them on endoplasmic reticula,
until it makes very, very large bodies.
Those bodies, then, reproduce themselves, move around the cells, as I'll show you later,
and eventually, then move from cell to cell.
And the real challenge here is for the virus to use the cellular machinery to do all of these processes
and to move from cell to cell.
Unlike animal viruses, plant viruses can't bud from their cell membrane and then be adsorbed by others
because plant cells are surrounded by very rigid cell walls.
These cell walls are penetrated by structures that are known as plasmodesmata.
This cartoon, developed by my colleague Bernie Epel, in Israel, shows a little bit of the structure of those plasmodesmata.
This represents a cell wall between two adjacent cells, that you see here and over here.
And going through that hole in the wall is endoplasmic reticulum.
Now, myosins and actins and other energy conducting structures
make modifications throughout the living part of the plant's life cycle
and allows this membrane to move from one cell to the next in a very metered way.
These structures maintain homeostasis. They're very small and only allow very small molecules to move through,
including ions and energy carrying materials.
If that's disrupted, of course, that changes the homeostatic nature of the leaf and it would be damaging.
There are in fact, during early parts of development and later parts of development,
you can see some changes in the structure of these plasmodesmata.
Here you see they're very much more rigid and they're bolstered by additional beta-1,3-glucans
and other molecules that give some rigidity to those structures.
So, the question is, how does a virus manage to squeeze through that structure?
And different viruses do it in different ways.
In this diagram, we've shown a diagram for how a set of viruses called geminiviruses
move from cell to cell. These are single-stranded DNA viruses, they replicate in the nucleus,
they come out to the cytoplasm, and one of the parts of the protein-coding encoded by the virus
moves it from the nucleus to the cytosol, and then another protein carries it all the way through the plasmodesmata,
as you see over here on the side.
Tobacco mosaic virus, by contrast, does it in a very different way.
There's an interaction between the movement protein, membranes and the cytoskeleton,
which carry these structures to, and through, the plasmodesmata.
And that's a subject of most of my talk this morning.
What we've done is use tobacco mosaic virus in a variety of ways.
We of course use the fluorescent protein green fluorescent protein as fusions to the movement protein,
sometimes as fusions to the code protein, to follow infection around the cell, and between cells.
In other cases, we've used simple confocal microscopy, coupled with immuno-localization assays, or procedures.
So, let's get into the nature of infection.
In this structure, in this study we've infected the virus into a protoplast,
these are cells from which we've taken the cell wall, and so that's why you see it as a round structure,
and we've identified the three different virus proteins with different colors.
The red represents the location of the virus replicase, using an antibody specific for the replicase.
The green represents the location of the movement protein, and the blue, the site of the coat protein.
Now, this is at about fourteen hours post-infection, and as that structure rotated,
you were able to see that some of these spots overlay each other.
What that should indicate to you is that they're in similar locations.
But as that went around, you know that at fourteen hours,
most of those structures were on the surface of the protoplast, on the outside.
In this case, now at 21 hours post-infection, now as you see this rotation,
you see that the green bodies are larger than they were in the first slide that I showed
and you see a little bit more overlap between the red and the green
and we'll come back to that a little bit later, that would indicate a co-localization of the replicase and the movement protein.
Notice that the structures that are green are much larger in this slide then they were in the previous.
This is at 21 hours after infection.
Now, a few hours later, at 26 hours post-infection, you see these very large green masses.
These are essentially the very large virus replication complexes, or VRCs, as we've called them throughout this talk.
And note again that there's a lot of overlap between the green and the red,
and the blue color, where the virions are assembled, tends to lie around the outside of these complexes.
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.
It's interesting to note that in this infection, the cell can produce as many as ten to the six virus particles per cell,
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,
allowing the cell to continue its normal metabolism and function.
We're looking at this now in a relatively early stage of infection,
but now cutting through the sections of these immuno-localized proteins.
And you see that you see a lot of green, again, the movement protein, located around the outside of the cell,
in these large replication factories, and in red, notice,
what surprised us in some of these studies, what in some cases you see mostly replicase in one section.
In other cases, you find the red and the green overlapping each other.
It's clear that the red is inside and the green is on the outside, as shown on this slide.
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.
And note that the red is sort of contiguous in some of these photographs.
For example, in this one over here, you see the red that goes all around that body.
We take that to mean that the red is on endoplasmic reticulum, which would of course have a lumen,
and the green would represent the movement protein, which is over top of the replicase.
The movement protein also itself interacts with the membranes in some cases, and in some ways.
So, what we've struggled with is understanding how the movement protein,
which is responsible for helping the virus move from cell to cell,
functions in building these replication factories, if at all.
And if the function in fact is to move it from the site of where these factories are located
over to and through these plasmodesmata, we're curious what the architecture of that structure is,
and we'll come back to that a bit later in the talk.
So, here's a thin section micrograph view of the virus replication complexes.
The arrows outline a series of structures that you see here, that's our interpretation of a virus replication complex
that you saw in a previous slide, as indicated by fluorescence microscopy in the confocal image.
Now, Dr. Katherine Esau more than fifty years ago described these rope-like structures that are found inside this complex.
Notice that there is not an indication that this complex is surrounded by membranes.
In fact, there's no indication at all that there are membranes except on the inside of the complex.
So then one wonders, how does a cell manage to wall off this thing so it doesn't take over the cell?
Or, how does a cell, how does a virus complex manage to escape the degradative enzymes that are present in the cytoplasm?
The RNases and the proteases, what keeps this all working together so that it makes more virions,
where you don't have disruption of either the virus replication complex, or severe disruption of the cytosol,
and killing of the cell.
It's especially interesting when one considers silencing of RNAs
and how the cell can defend itself against intruding pathogens, such as viruses,
how does this manage to keep out the silencing mechanism so that it doesn't degrade the pathogen in the process.
In fact, the host has very little defense against this kind of virus.
This is now a close-up view of these structures, and notice these rope-like structures.
So, what are they?
And there are, if you look carefully, there are other membranes.
And more recently, we have used immuno-gold localization to locate the proteins
and sure enough, on these structures, on these rope-like structures, one finds virus replicase and some movement protein.
But what's the structure of them?
And recently, we've worked with colleagues at the University of Chicago
to begin to tear this apart by using tomography.
And I don't have all the data are not in yet, but eventually,
as you know, a tomogram is developed on relatively thick sections that are produced for electron microscopy,
in the standard fashion, and then uses a high energy beam to itself slice farther down into that section
than is seem in normal transmission electron microscopy.
And then the section is tilted very slightly, at one or two degrees at each time,
and then a new photograph is taken.
And from that, one builds an image. Now, we don't have the work finished yet,
but I wanted to tell you where we are to date.
So, we have been working towards creating a tomogram
and of course, using these tilting stage and collecting vast amounts of information,
we then compile that information into a three dimensional image that tells us more about how the structure works.
And this is just a preliminary, but you can see where we're headed.
Note these rope-like structures that you see here; that's like what I showed you before,
but since this is now a tomogram, and it's a non-stained section, it's a little less distinct.
But you can see, over towards the middle of this slide,
this continuous area that we have outlined in a very light green color,
and you see that's what we represent as the endoplasmic reticulum.
So, inside of this lies the lumen and on the outside, this is the outside structure of the endoplasmic reticulum.
Now, if you recall your cell biology, there are rough endoplasmic reticula and smooth endoplasmic reticula
and in fact, these represent ribosomes on this one, so this would be a rough ER.
When I was a post-doc nearly 35 years ago, I was curious about how viruses are assembled
on these membranes and finally, we were able to see it using the new tools of high energy electron microscopy and tomography.
Now, the structures that are indicated here in this other color, kind of a magenta color,
are ribosomes that link the ER and the rope-like structures.
Now, if we were guessing, and we are simply guessing right now,
we think that these are the links that hold the ER and the replicase and the ropes all together.
And they're in a structure that, with this linkage of rough endoplasmic reticulum and poly-ribosomes and replication,
all happening on the same complex that builds into this rope-like structure.
We hope over the next several years to be able to tell you more about these complexes.
They really have given us an insight as to how these complexes are built.
That's one side of the challenge.
The next side of the challenge is how does this thing move around the cell and between cells,
if in fact it does.
Now, what we see here is an image of an infected plant leaf.
Now, this is a transgenic plant in which the actin is labeled also with talin.
So, you can see the fluorescence of the actin cytoskeleton.
Up here, of course, you can see the location of a stomata, the holes through which oxygen enters,
and gaseous exchange occurs.
Here you see a non-infected cell. And down in this section you can see in fact that there are these globs,
these blobs that I talked about earlier.
Those represent the virus replication complexes that we talked about before.
So, what we're looking at is a very interface between an infected cell and a non-infected cell,
this one being non-infected,
and these down here being infected.
Notice that there is not, in addition to having these blobs, or virus replication complexes
in the cytosol, there are also some that are tightly oppressed to the cell wall.
The position near the cell wall represents where the plasmodesmata are.
That we know by electron, other electron micrographic studies.
And so this front shows that in this cell, at this point, is not heavily infected
but yet there are still some plasmodesmata that are labeled with the movement protein
and the replication complexes, either in or adjacent to the cell wall.
In other cases, you can see that these factories are still out in the cytoplasm.
This would represent where most of the virus replication is happening early on,
or at least where most of the movement protein is being made,
and then in this cell, it's a younger infection and this cell is a non-infected cell.
Now, if we put this into some sort of imaging, into a time frame,
you can see that these globs move around within the cell,
and they fuse together, you saw that one just fuse with the large one, the small one fuse to the large one.
This large guy is going to start moving down on these actin cables as well and moves all around the cell.
That movement is very dynamic and we can mark it by simply putting the microscope on a cell
and plot the rate at which it moves by simply measuring the rate of movement in real time.
So, in the next slide, I'm going to show you a series of stages of infection.
One will be at 18 hours, at early after infection, 14 hours, and then 16 hours and 18 hours.
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.
And then the 16 hour stage, the bodies are moving very, very slowly.
By 18 hours, they're dead stopped.
And then nothing happens for a couple of hours and then I'll show you, down in a lower portion of the slide,
a section in which the virus infection has moved from one cell to the next.
So, again, up in the upper left-hand corner, that's where the infection is now only 14 hours old
and you see those bodies are moving very, very rapidly, as indicated by the number below, 116 nanometers per second.
At 16 hours after infection, those bodies are moving more slowly.
Very slowly indeed.
By 18 hours, they've essentially stopped.
And then at 20 hours, two hours later, you now see the infection is not just in a single cell,
but is in three cells. The virus infection has moved from the first cell out to the next.
And now the cells that are recently infected, those that are up here in the one portion
and those that are down in the lower portion, are moving very rapidly.
They're in fact moving at the same rate as the bodies at the 14 hour infection.
What that told us was that in the first infected cell, it takes a long time to set up the virus replication factories.
They start, they're made in that first 6 to 12 to 14 hours,
by 14 hours, they begin to move around and fusing with each other and moving to the cell walls.
And then they stop moving, there's an immobility to the whole, to the whole process,
we had noted this before, that there's a time frame in which the mobility of cytoplasmic bodies stops.
At this point, these, you have the replication factors against the cell wall
and then we have other films that show the virus bodies, these replication complexes moving from one cell to the next.
That surprised us.
It was the first indication that what was moving from cell to cell was not the virion,
but some sort of a pre-virion complex,
perhaps containing viral RNA, maybe even double-stranded RNA,
and replicase and other components of the factory that were necessary to get it all started again.
Then it goes into the next cell
and the replication cycle doesn't have to start from zero.
It now can start from having built up, from that first complex that was sent over.
In fact, by, if the first infection requires 20 hours to move from the first cell to the next,
going from cell number two to cell number three to cell number 4 is about an interval of four hours each.
So, it moves very rapidly from cell 2 to cell 3 and that's why the virus infection can move throughout the cell,
and throughout the tissue, throughout the whole plant, in a relatively short period of time.
It moves between cells, not as virions, but as sort of pro-virions, or pro-structures that include the replication complex.
What holds all this together?
We've been looking at the structure of the movement protein and asking how it can work.
It appears to be an integral membrane protein.
It has a trans membrane domain that's fixed here,
if we make mutations in those amino acids that are in the membrane portion, as indicated by the red bubbles,
we changed the localization and it no longer integrated into the membrane.
We do not have a structure of the movement protein
but this is a good representation, at least where we are today.
On the cytoplasmic side, as you see up here,
these are the cytoplasmic side where both the N-terminal and the C-terminal parts of the protein are exposed,
there's also a myosin binding site that a former colleague has identified.
Those, that might indicate that by binding here, in this region, with the myosin and linking to actin
begin to, helps to tell us how these structures move around and through the cell.
After these years, and we've been looking at this for a number of years,
we have the following picture of how tobacco mosaic virus as an example of a plant virus moves from cell to cell.
In the early stages of infection, as I indicated earlier,
the virus encodes these proteins that are replicase and movement protein
and in some cases, we can see that they go to different sub-sections of the membrane structure of the cell.
Those then collapse back onto the nucleus, where the first set of the replication happens.
It then moves out into the cytoplasm and builds large replication factories,
some of which are shown here in these big globs.
Some of these, but not all, will bind to actin-myosin filaments,
and then move around the cell. In that process, many will move over adjacent to the plasmodesmata.
Now, the question remains how these bodies might move from here to here,
a colleague Bernie Epel in Israel has indicated that that movement is driven by a variety of motive forces
which help to move proteins between cells. It's a very curious and interesting process.
But yet, one has to imagine that the architecture of these walls also change.
Now, these are very rigid cell walls, so there must be some softening of the walls,
with enzymes that are carried perhaps on the same complexes that create the virus replication complexes,
extruding those into this space between the membrane and the cell wall, softening it to the point that it opens and closes.
Working with a colleague, Karl Oparka, nearly ten years ago, we found that these holes,
these plasmodesmata open for a short period of time at the very leading edge of the infection site
on the inoculated leaf, or the infected leaf.
So, it opens and then allows this to happen, to transmit, and then the hole closes again.
That's in line with the importance of maintaining a homeostatic situation within and between the cells.
So, that's where we are in the model.
There are lots of questions that remain and we and others have been looking at it,
though it's not an easy, the questions are not easy to address.
For example, how does a replicase work with a movement protein in the ER, in interactions that create the virus replication complexes,
and move them around the cell.
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.
Certainly there have to be host proteins involved and from a virology standpoint,
one suggests that the sites on the endoplasmic reticulum are kind of internal receptors,
the places inside the cell where virus proteins go, where they know where to locate,
perhaps because the nature of the membrane itself or because of proteins that are embedded in the membrane.
The nature of those will take a proteomics approach for analysis and then some genetics to try to interfere with the process of docking.
Once we know more about those structures to which the replicase and the movement protein dock,
we'll know more about these internal cellular receptors, perhaps they can then be targets for blocking
or they will tell us more about the assembly of these factories and then that will tell us how they move around the cell.
How they attach? We don't know.
And then how do these attach to the myosin-actin fibers for transport to and through the plasmodesmata,
what's the process of opening and closing, and essentially, how does a cell manage to live through this process,
where the structure is re-oriented and reorganized, making perhaps as many as ten million virus particles per cell, and not killing the cell.
What is this compatibility that happens in pathogenicity for this virus?,
are some of the questions that we're addressing.
In the subsequent lecture, I'm going to talk about how we use this information to develop genetic strategies,
or genetic engineering strategies, to help the plant block some of these processes
and then thereby developing virus-resistant plants.
Some of these are crop plants that will be useful for producing food in agriculture settings.
So, I hope you'll join me for the second lecture and I'll sign off for now and leave you with these questions.
Perhaps you will be the one that will answer what some of these questions in the future. Thanks.
