AUSTRALIAN FRONTIERS OF SCIENCE, 2008

The Shine Dome, Canberra, 21-22 February

Session 2: Discussion

Barbara Howlett: You have heard now about two fungal diseases of wheat. In the first one, the fungus needs to keep the plant alive, and yet in the second one we have got the fungus killing the plant – indeed, having to kill the plant for the fungus to be successful. Another point that we think about is the evolution. So, in the first case, we hear about the co-evolution, the selection pressure that varieties with resistance genes put on the fungal population, and in terms of evolution a very dramatic single-gene transfer effect to, in fact, cause a new disease.

Question: That is really amazing, to have this gene, ToxA, crossing from one fungal species to another. Do you know where in the world it happened, whether there were special conditions that allowed it to happen, and whether you can stop things like that from happening again in the future?

Peter Solomon: Where in the world? We are narrowing in. We think it is probably in the Middle East somewhere.

Special conditions? I don't know. I think there is a high possibility that it is just a chance of fate: they happened to be in the right place at the right time, and probably underwent an event known as anastomosis, where two hyphae just fuse together.

Can you stop it from happening again? No, you can't. Something I would like people to take away from this is that no, you can't stop it. We talk about avian bird flu and things like that: these things can jump, they really can. It can take just one gene. In this case it was fungus to fungus, but it was just the one gene that caused the change. No, I don't think you can control it.

Question: Leading on from that: are there any other cases that are known for a disease like this to have happened as well?

Peter Solomon: Yes. I think it is fair to say that there are, and Barbara might like to comment on this as well. She has worked on horizontal gene transfer as much as I have, in other fungi. I think it is also fair to say that there is nowhere near the level of evidence that we have got. We have been very lucky as far as resources go, with strain collections, good collaborators and things like that.

Barbara Howlett: There is lots of evidence of biosynthetic pathways and clusters of whole genes moving around and, as Peter showed, the evidence of the transposable elements, the 'jumping genes', nearby. But I guess one of the very exciting things about what Peter has told us is that it is really just a single gene that has had such a dramatic effect.

Question: There seemed to be some uncertainty about how these rust proteins get into the body of the cell and then into the nucleus. If you could figure out how that happened, would it be fair to say that that is an alternative approach, that you could engineer some method of blocking that transfer?

Peter Dodds: I guess it depends on what the transfer method is. One possibility is that each of those proteins has a different mechanism, and they may be proteins which are intrinsically able to cross membranes, in which case there is probably no mechanism that you could target. The alternative is that there is some specific transport mechanism that recognises all of those proteins and just picks them up and puts them onto the other side of the membrane. And then in that case, I guess, yes, that is a target you could use for engineering resistance.

Question: I also have a question about the rust effectors. You talked about inducing mutation in some of the known resistance genes. I am wondering if there is some strategy for how you do that. Is it completely random? Is there some guiding principle in trying to define ones that match what you are looking for?

Peter Dodds: In the experiment that I am talking about there, where we want to just try and engineer a resistance gene, we just want to do it at random. We just want to generate random mutations, usually PCR-induced random mutations, so we have got a large collection that we can select from.

Another thing we are working on at the same time is to use the model we have for how they may interact, to try and predict which of the particular amino acids are involved in that interaction, and whether, if we change them, we can change the recognition specificity.

That is an approach which is experimentally useful to understand the science of it, but if you want to use it to engineer something, you don't want to be tied down to having to have that really high level of information about how they interact. You just want to say, 'Well, we know this interacts with this. Let's just change it and select what comes out and is useful.'

Question: Just moving on from a previous question: is the invagination process of the haustorium something that would require an active plant role in that, some sort of almost pseudo retrograde transport as well, in invagination as well? And is that an area where we could look at controlling, certainly, rust fungi?

Peter Dodds: That process is really not understood. A lot of things have to happen there. As the fungus grows into the plant, the plant membrane itself has to grow around the fungus, so there is something happening on the plant side as well that is involved in that process. Whether or not that is a modification of a normal vesicle uptake mechanism which has just spread out to make a really large vesicle, we really don't know – nor how much of that depends on specific plant factors and how much of it is induced by the fungus itself. So we don't know much about it.

Question: I have two questions. The first is for Peter Dodds, in regard to introducing mutations. You showed that interaction between the resistance gene and the effector molecule is based on structural interaction. By introducing the mutations you will be destroying that structure to some extent. How are you going to overcome that?

The second question is for Peter Solomon. You said that ToxA has to be there. But is it affecting the pathogenicity of the fungus itself? As a target perhaps to avoid it, is anyone – including yourself – looking to maybe getting specific inhibitors of ToxA?

Peter Dodds: In the structure of the resistance protein, that curved structure which is the recognition domain, there are residues which are important for the structure. If you mutate those, you are going to knock out the structure and it is just not going to work. The residues that are important for recognition are the ones that are exposed on the surface, and we already know that the protein structure can tolerate an enormous amount of variation in those positions, because if we look at the variants that exist already we see enormous variation – but only at certain positions in the protein. And they are the positions that encode amino acids that are actually sticking out from that protein into the medium. So we think we can generate a lot of mutations at those sites which don't affect the structure but will affect the recognition.

Peter Solomon: The experiments I put up on the board were showing interactions between ToxA and the corresponding resistance gene in the plant, Tsn1. Although I haven't talked about it, the reality is that Stagonospora nodorum produces about 13 of these things, and has 13 corresponding genes in wheats. I mentioned that this disease was a quantitative disease, and there are different parts to it. We know that the ToxA–Tsn1 interaction in most wheat interactions contributes to about 20 per cent of the disease. So if you have a wildtype fungus with all its toxins and a wildtype wheat with all its dominant susceptibility, that particular one interaction is a reasonably serious one. It contributes to about 20 per cent of the disease. We don't know the other genes yet – I am trying to secure funding to work on that.

As to targeting ToxA to design specific inhibitors: definitely. The research groups that have been working on ToxA in Pyrenophora have been doing so, clearly, for a lot longer than we have – as I say, we have really only just identified it. They have crystallised it, et cetera. The quickest way round it for us to protect our wheat against our fungal diseases is to try and look for wheat lines that don't contain the Tsn1. It is amazing, when you actually go and have a look at wheat that we eat, wheat that we grow commercially, that it nearly all contains Tsn1. To us that seems counter-intuitive, but things are there for a reason. That Tsn1 must be there for some reason. We don't know why yet; this thing just renders it susceptible. As for removing it, we can't see a great effect in similar wheat lines that don't have it, on yield or any other factors such as that, but there clearly must be or it wouldn't be there.

So yes, we will target the ToxA, I have no doubt about that. And, secondly, as far as crop protection goes, at this stage let's look for things that don't contain Tsn1.

Question: I have got a related question for Peter Dodds. Your engineering approach sounds very similar to phage display libraries, with synthetic antibodies. Those libraries can have 109 or more different antibodies. So my first question is: how big is your repertoire of mutations that you can create with PCR? The second question is: why do you use yeast? And the third one is: can you use phage display for your approach?

Peter Dodds: Let me start with the third part: can we use phage display? I don't know. I don't know how well these proteins will express under those conditions. We are planning to use yeast for that because we have been using the yeast-to-hybrid system extensively to analyse the interaction between these proteins, and so when we make mutations at single sites in the rust effector protein we can see differences in the yeast-to-hybrid assay which exactly correlate to what we see when we put the same proteins onto a plant and induce a response. So we know we are looking at the same quantum of interaction: if we measure a certain affinity in the yeast-to-hybrid system, it corresponds to what gives the response in the plant. That is the main reason for using that system.

As to the number of variations, I think it is probably not to the same extent as you can get with a phage display for antibody work. But I am not quite sure how high we can go there. I think we can probably get to 106 or 107.

Question: I am blown away by the fact that such a small random occurrence somewhere can have such a massive effect. It feels to me that there is an element of chasing our tails on this. These effects are studiable and take many years of research to get to the bottom of, but looking at the history, you know that in decades to come there will be more and more specific, individual cases that need to be followed. So can you comment on the underlying development of the body of knowledge that is going to make the future better in this regard, or is it always going to be that in 50 years' time we'll be talking about the 'effect that arose in 2010' type of situation?

Peter Solomon: My thought is that genome sequences are going to rapidly advance this field beyond belief. I think evidence of that has already started to come through from our work and others' as well. That genome sequence has shown us that this protein is present; we now know that many others are present. The beauty of this is that if one protein in the fungus equals one protein in the plant, we can start selecting against those. Okay, we might then start to run into problems such as Peter Dodds is talking about, with resistance and the genes themselves within the host evolving, and things like that. But I would like to take a slightly more optimistic view, as I guess we all do, that what we are doing now is like a logarithmic expansion – we are getting to that point of, 'Yes, we are going to make a difference.' And we are making a difference now. Everything we have done to this date has made a difference. We are getting there. But no, I'm not working for 50 years' time.

Question (cont): So you can basically attack these things and solve them faster…

Peter Solomon: Yes. After the work alone that we have done with this, we have already gone to the farmers and said, 'Look, you don't want this protein. You don't want this gene here.' We've gone to the breeders and said, 'Screen for this thing. Don't have it.' And they are actually listening to us for a change. So it's good.

Peter Dodds: I think the other thing in terms of our disease system where we are looking at a resistance, a recognition mechanism, there are two things we can think about to try and generate more durable resistance.

For the moment what we have been relying on is just whatever has evolved in nature. You pick up some resistance genes that you put in, and they are very effective and last a long time. And you pick up others, you put them in and next year they are gone. So we are really just getting to the beginning of this. We need to understand what it is that makes some more durable than others, and I suspect it is to do with how valuable the effector protein is to the pathogen. So we need to be able to target things which we think are really important to the pathogen, such that it just can't change them easily.

There is another thing we need to do. (Often in breeding we try to achieve this, but it is not always possible.) You find that you put one resistance gene in, and the pathogen has only got to change one effector protein to escape resistance. If you put in five, the pathogen has got a really hard time because it has got to change five things at once to be able to overcome resistance. So that is the other strategy. One is to target things it can't change easily; the other is to target lots of things at the same time.

And again the genome sequencing resources that are coming out are starting to make those sorts of numbers available, so we can do that.

Barbara Howlett: These studies really emphasise the co-evolution of the pathogen and the plant. The fungi are always changing. The breeders try and keep up with it. But I am, like the two Peters, pretty optimistic, particularly with the genome sequencing being available.

Question: On the subject of co-evolution of these genes, I was wondering whether plant genes like Tsn1 could have evolved in terms of interactions with other fungal species that are perhaps beneficial to the plants, because there are lots of beneficial interactions between plants and fungi as well, and whether research into knowledge of the beneficial interactions between fungi and plants could lead to some better insights into the pathogenicity and interactions of genes like Tsn1 and ToxA.

Peter Solomon: That is an excellent point. That is entirely likely. It baffles me why a plant would harbour a gene that renders the plant itself susceptible to a disease. And yet we do think it is something like that. The current working theory is that it is a hypersensitive response gone wrong. Essentially, we seem to think that our fungus is tricking the plant into thinking, 'Right, create a hypersensitive response' – similar to what Peter Dodds was talking about. But instead of being in a situation where that is going to kill the fungus off, ours likes it. It has essentially just tricked the plant, and says, 'Right, I've got you now.' That's one line of thought we are working on at the moment, and I have no other lines of thought so I am going to stick with that one. It is a good question, though.