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Univeristy of South Carolina s Vicki Vance Discusses RNA Silencing


At A Glance

Name: Vicki Vance

Position: Professor, University of South Carolina

Background: Associate professor, University of South Carolina — 1995-2000; Assistant professor, University of South Carolina — 1989-1995; Research assistant professor, University of South Carolina — 1988-1989; Postdoc, University of South Carolina — 1983-1988; PhD, plant biology, Washington University — 1983; MS, microbiology, University of Illinois Medical Center — 1973

After spending a few years as a virologist and bacteriologist, Vicki Vance entered the world of plant biology only to find her work extending into a field of post-transcriptional gene silencing. Recently, Vance spoke with RNAi News about her research and its role in RNA silencing.

How did you get involved with RNA gene silencing?

The way I got involved is that I was a plant virologist and I was trying to understand mixed virus infection in plants. A lot of times plants are infected with more than one virus at the same time [and] sometimes the two viruses cause a much worse disease. I was interested in understanding how that worked.

That’s called plant viral synergism, when you have these two viruses that interact and cause a much worse disease. There are lots of plant viral synergisms and what was interesting was that in almost every case, one virus in the pair was from a group called potyviruses. So it would always be like there was a potyvirus and some unrelated heterologous virus, and the two would cause a much worse disease. In every case anyone looked at, they would see that the non-potyvirus of the pair would accumulate to a much higher level. It was like there was something about these potyviruses that could help a broad range of unrelated viruses to accumulate to a higher level, and this was occurring in a broad range of crop plants — and also probably in nature.

I wanted to understand what it was, so we took a transgenic approach where we expressed various potyvirus genes in tobacco, and then looked to see if you needed the whole potyvirus or just one or a subset of the potyvirus genes. That research is what dropped me into the middle of the silencing field because what we found is that you don’t need the whole potyvirus and that there was this protein — called helper-component proteinase, or HC-Pro for short — [that] by itself could help all these viruses. How could it be doing that?

What we hypothesized is that HC-Pro helped all these different viruses accumulate to a higher level by interfering with the general virus defense mechanism. There was no general viral defense mechanism in plants known at that time, but people were beginning to think that maybe post-transcriptional gene silencing was just such an antiviral defense pathway. We hypothesized that HC-Pro was interfering with post-transcriptional gene silencing. That was a testable hypothesis because there were all of these transgenic plants that were silenced for some reporter gene and we had transgenic plants that were making HC-Pro. So, we just crossed them and looked to see if the offspring were still silenced. What we found was, indeed, HC-Pro could block transgene-induced post-transcriptional gene silencing.

It was really exciting. Here was a viral protein that was interfering with [what was] at that time a very poorly understood phenomenon. It was going to be an important tool to try to understand how the whole thing works. And that’s what we’ve been doing — trying to understand where HC-Pro blocks silencing and how it blocks silencing.

Can you touch on the projects you have ongoing and how you’re trying to answer this question?

We looked at various aspects of RNA silencing to see what would happen when HC-Pro suppressed silencing, and we’ve been using well-characterized, stable transgenic lines that are silenced by different kinds of transgenes. What we find in every case is that HC-Pro blocks the accumulation of the siRNAs, the small RNAs that act as guides to mediate sequence-specific RNA degradation. It also enhances the accumulation of the endogenous microRNAs. The siRNAs don’t accumulate, but in some cases long double-stranded RNA does accumulate in the HC-Pro plants.

Since the product of Dicer goes away and the substrate of Dicer accumulates, we think that HC-Pro is altering Dicer activity. So, we looked at microRNAs, which are also made by a Dicer — in plants there are four Dicer-like genes — [and] what we see is that microRNA accumulation is changed, only it is increased. We think that HC-Pro is altering Dicer activity in a kind of complex way that I think is going to turn out to be pretty interesting.

One approach has been to look at these various biochemical aspects of silencing and see if they are changed or not by HC-Pro. We’ve found that small RNAs are changed, the siRNAs go away and the microRNAs increase, methylation of the silenced transgene is unaffected by the HC-Pro, and the ability to send the mobile silencing signal is unaffected. So, HC-Pro blocks the silencing but doesn’t prevent either transgene methylation or the mobile silencing signal.

We want to try to understand how HC-Pro is working, so one thing we looked at is other defense pathways. It looks like silencing is a defense pathway and HC-Pro blocks it, so we wondered if HC-Pro could also block some other known defense pathways. We looked at resistance to tobacco mosaic virus through a resistance gene called the R gene [because] we thought that the HC-Pro plants could overcome that too. Instead what we found is that the HC-Pro plants had enhanced resistance, and they also turned out to be more resistant to a broad range of other pathogens. So it seems like HC-Pro blocks silencing but it’s potentiating a different defense pathway that’s mediated through salicylic acid.

Another thing we’re doing is looking at how HC-Pro might work by looking at plant proteins that might interact with HC-Pro. We found a number of proteins that [do this], and then we were trying to over-express those proteins or block their expression to see if [HC-Pro] affects silencing. By doing that, we found a calmodulin-related protein that, when over-expressed, also suppresses silencing. So, we’ve been trying to understand how that calmodulin-related protein works and what the targets of that calmodulin-related protein are. Presumably, it’s part of an endogenous regulatory system.

We’ve been working on that for several years.

Can you speculate on the long-term implications of your research — where it’s headed?

I guess I didn’t mention anything at all about the practical aspects of our work. A lot of plant biotechnology people would like to be able to over-express genes in plants — some beneficial genes — and it seems that silencing is a major problem. It looks like you can over-express something to a certain level but it’s almost like you reach a threshold, and what happens if the transgene silences? We’ve been able to use HC-Pro to directly block transgene silencing so that you can get high-level, consistent expression of beneficial transgenes in plants.

There are several patents for practical uses of HC-Pro and I think those have had a pretty big impact. There’s one modification where we use an amplicon transgene, which is a transgene that makes a viral RNA that then is a vector for whatever protein you are trying to express. So here’s a transgene that’s present in every cell, it gets expressed, and then it replicates. And it replicates through double-stranded RNA intermediates. Initially, people thought these amplicon transgenes would give extremely high-level expression, but instead they found that every single primary transformant was silenced. In retrospect it makes sense because they’re double-stranded RNA, so it turns out that the amplicons are very potent inducers of silencing.

But HC-Pro can block that amplicon-induced silencing, then you get the earlier-envisioned very high level of expression. People are trying to use that to make pharmaceuticals or antibodies in plants, so it makes a real big difference to have a much higher yield when you’re trying to purify those things. A number of companies have licensed this patent, called amplicon-plus.

This patent is from your research?

Yeah. I’m the inventor on that patent, and it’s licensed to a company called PBL — Plant Biosciences Limited — and they handle all the intellectual property. They sublicense [the patent] to companies to use.

For us, that’s been really great because we’ve been able to get grant money from companies to basically develop this technology that we’re trying to develop anyway. We’re not really interested in the practical aspects, but we’re interested in how it works and these people give us money because they want to use it.

In terms of projects down the road, are there any areas in this field that you’d like to tackle, or is there [related research] that you’re closely watching?

Of course, we’re really interested in the whole microRNA field, in part because HC-Pro affects that pathway in a rather complicated way. It looks like it increases the accumulation of the mature microRNA, but it blocks their function — at least in some cases it looks like it’s blocking their function. So, it looks like it may be working at the level of Dicer that’s making small RNAs, but also downstream in the RISC complex. Maybe that’s not too surprising with this more recent data that Dicer may be involved in both. So, we’re real interested in microRNAs and there’s just so much to be done with them. It’s such a big field and there’s so much to do.

It’s just getting started so it’s really fun to be involved in a field like that. Everything is wide open and it’s almost like anything you do [you end up] finding something really interesting.

We’re working with Dow right now to try to get HC-Pro suppression of silencing in transgene-expression corn, and the corn people are real fun.

I’ve never heard that before about corn people.

They are, they’re fun. It’s a nice little group of people and they’re all very positive about one another. A lot of the silencing field and the microRNA field is little bit competitive and a little cut-throat, so a little foray into the corn world is lot of fun.

The other thing I’d probably go back to are these plant-viral interactions. I figure that the whole silencing/ microRNA field is so competitive and … of course the animal people are just coming in … so I’ll probably go back to plant-viral interactions. We have some really interesting ones — we have a plant-viral protein that looks like it interferes with this other defense pathway, the salicylic acid-mediated pathway. We’re pretty excited about that, and that’s kind of a fall back for when I get squashed like a bug in the RNAi field.

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