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USDA s Bret Cooper on Using Mass Spect to Study Bean and Soybean Rust

Bret Cooper
Research plant pathologist
Soybean Genomics and Improvement Laboratory, US Department of Agriculture-Agricultural Research Service

At A Glance

Name: Bret Cooper

Position: Research plant pathologist, Soybean Genomics and Improvement Laboratory, US Department of Agriculture-Agricultural Research Service, since 2004.

Background: Staff Scientist, Plant Health Department, Torrey Mesa Research Institute, 2000-2003.

Associate scientist, Novartis Agricultural Discovery Institute, 1999-2000.

Postdoc, Department of Cell Biology, Division of Plant Biology, The Scripps Research Institute, 1998-1999.

Postdoc, Departments of Entomology and Plant Pathology, University of California, Riverside, 1995-1998.

PhD in plant pathology, University of California, Riverside, 1995.

Bret Cooper gave a talk on "non-model species proteomics" at last week's Association of Biomolecular Resource Facilities conference in Long Beach, Calif. ProteoMonitor spoke with Cooper after the conference to find out more about his background and work on bean and soybean rust.

How did you get into investigating plant pathogens using mass spectrometry?

I've been a plant pathologist for much longer than I've been a mass spectrometrist. I got a PhD in plant pathology. I'm not sure why I went into the field, other than because I was interested in the technology of making transgenic plants that could be resistant to diseases. During some of the early part of my career I dabbled with trying to make plants resistant to viruses and other pathogens. They were tobacco plants — model systems.

The first paper I published was demonstrating the expression of proteins in plants that would give plants resistance to many different viruses. I was using transgenic technology — taking foreign genes and putting them into plants.

I think what comes from all of that work is this need to identify the pathogen in the plant. So if you make a transgenic plant, and you think it's resistant, you have to be able to measure that somehow, right? You have to be able to detect the pathogen, and see how much or how little is there to see if the technology is performing very well. So every plant pathologist needs to able to identify his pathogens. So I've always been aware of the different types of pathogen-detection capabilities, and known their limitations.

One limitation has always been this requirement of needing a pathogen-specific reagent, or a pre-formed reagent for detection. So if it's an ELISA test or a Western blot, you have to have an antibody already made to the thing that you want to detect. If you want to do PCR, you have to know what the sequences are for the pathogen. For many plant pathologists, getting those resources can be difficult.

I've used all types of detection methods. I've used host-range studies and looked at symptoms on different plants. You can do crude types of purification. For viruses you can do centrifugation, for fungi and bacteria you can grow things out on plates and look at them under microscopes and try to identify them. Then you move in to more practical molecular approaches like Western blots and PCR and DNA sequencing. For viruses there's even a procedure called double-stranded RNA analysis that was always pretty rigorous for identifying plants infected with viruses.

We used all of this stuff, and I actually ran into a problem a few years ago when we were doing some studies on plant responses to virus infection. We were using a virus that basically came out of a refrigerator from a very old collection, and the virus didn't behave like the name on the tube. We wanted to publish this data, but we weren't confident that the virus was actually what was named on the tube. So using the information on the tube, we tried to design PCR primers to identify this thing, and we just couldn't. We did some host-range studies, and they weren't really conclusive for the identity of this particular virus.

I was pressed for time, and I was friends with some mass spectrometrists who were evaluating plant proteins, and I asked them, 'Do you think you could identify the virus proteins among all the plant proteins?' They were able to, and were able to characterize this new virus for me. And that set off the whole program of trying to use mass spectrometry to identify pathogens and pathogen proteins for me. Necessity was the mother of invention here.

So after that, you started to use mass spec to find the pathogens?

Right. From that came a broader proposal to not just use mass spectrometry to identify viruses, but also to identify more complex pathogens in plants, like fungi and bacteria. We set about acquiring different fungal pathogen specimens — some that have a sequenced genomes, and others for which very little genome information exists. We began preparing proteins from those cultures and doing MudPIT-based proteomics on those samples to identify the proteins there.

What we've discovered is that it's easy to identify pathogens whose genomes are sequenced; much more difficult to identify pathogens whose genomes aren't sequenced. We still are dependent upon a sequence database to identify proteins this way.

We believe there are still uses for mass spectrometry to identify pathogens, and we are looking at those. Essentially, we want to just use the tandem mass spectra themselves as fingerprints, or biomarkers, for the pathogen, and get away from using the database search algorithms that can lead to misleading identifications for pathogens.

What did you learn by doing proteomics on these pathogens?

Well, one of the initial proteomic studies was on Uromyces appendiculatus, which is bean rust. We were able to identify some of the most abundant proteins in the spores, which appear to be heat shock proteins and translation factors. From this data, we've been able to propose a model that help support the theory that these spores are able to survive environmental extremes and produce proteins very quickly to infect plants in a short amount of time.

Do you think this information can help prevent an outbreak of the rust?

Not for that particular experiment. The initial experiment was for a study of resting uretospores, and now we are looking at the germinating uretospores. The germinating uretospores have proteins on the outside of the germ tube. Through a collaboration with Jim English at the University of Missouri, he has discovered that he can inhibit proteins on the outside of these germ tubes and essentially stop the progression of germination. So we're currently using mass spectrometry to absolutely identify the proteins on the outside of these germ tubes. So yes, I think the next round of mass spectrometry over this year will identify some targets that can be used to protect plants against diseases.

Is Uromyces appendiculatus the primary pathogen that you're working with?

Yes, at this time it's the primary pathogen. We want to work with soybean rust. There's a difference between bean rust and soybean rust. We have been working with bean rust because until January of this year, we didn't have a permit to work with soybean rust. We just got our permit, and we are making plans to work with soybean rust.

We will continue to work with bean rust, as well as soybean rust, because we want to study the bean rust/edible dry bean interaction because the edible dry beans have resistance genes that are very durable. Perhaps one day those genes, if we can get them, can be used in soybeans to protect soybean plants. That's a very big long-term program.

Do you have to have special safety laboratories to work with these pathogens?

We are permitted to work with each organism. Soybean rust permits are a little bit more stringent. Right now, unless soybean rust is in your state, you are not allowed to bring it in from another state. Essentially, you can only work with the isolates that are in your state. If you don't have any, then you have to have a special quarantine facility to work with the organisms.

We have the equivalent of a biosafety level 3 containment greenhouse for working with soybean rust. That is state approved. It keeps everything trapped inside. It's a pretty amazing facility.

The soybean … is the second largest crop in the US. The edible dry beans are ranked ninth among US vegetables, according to the US Department of Agriculture.

Is the main reason you're working with bean rust because it is similar to soybean rust?

I guess there are three main reasons. One: I'm not under the same restrictions that I am with soybean rust, so that makes it easier to work with. Bean rust is native to North and Central America.

Two: Is what I told you before, that bean rust has resistance genes that could be used in soybeans.

The third thing is the similarity between how the rust pathogens behave on plants. Basically, if we can work with common bean rust, we can work with soybean rust. The principles for plant pathology and infecting plants, and the equipment that we need are all the same.

When people come to me and say, 'Bret, I'm interested in working with soybean rust. What do I do?' I tell them, 'Well, you go get yourself a nice friendly strain of common bean rust and you start there.' Because you don't have to work in quarantine, and you can ramp up and learn about rust biology there very easily.

You were doing MudPIT experiments on the bean rust?

Yes. We've also done some MudPIT experiments on Ustilago maydis, which causes corn smut; Fusarium graminearum, which causes wheat head blight; Rhizoctonia solani, which causes seedling damping off; and Phytophthora sojae, which causes soybean root rot.

Did you identify key proteins for all of these?

Oh, I wouldn't go that far. For the pathogens whose genomes are sequenced, such as Ustilago maydis, we have had little trouble in identifying proteins using mass spectrometry. As for the biological significance of these proteins that we've found, we haven't evaluated that yet.

But for the other fungi, such as Rhizoctonia, we're having an awful lot of trouble identifying proteins in this fungus, probably because there is very little sequence representation for this fungus in the databases. And we aren't getting very good cross-species identification matches using proteins from other organisms either. So some of these fungi appear to be very unique in their protein sequences.

How can you progress without the sequence?

More and more fungi are being sequenced. I'm not involved in any of the pathogen sequencing projects. Other people are doing that, and they come along in time. I guess we have to wait for the databases to get better to identify some of these proteins for the more obscure fungi. Nevertheless, like I said, we are taking a database-independent approach to identifying pathogens by mass spectrometry.

So there's a difference between identifying a pathogen using mass spectrometry, and identifying a pathogen protein using mass spectrometry. We think that our biomarker approach that doesn't rely on sequence databases can be used to identify pathogens by mass spectrometry.

For identifying pathogens, we only are concerned with the spectra itself, not really with the final amino acid sequence. This type of approach has already been successfully used for the identification of small molecules. Really, it's the industry standard for identifying molecules based on their mass spectra.

Where do you plan to take this in the future, now that you've identified some of the proteins in these pathogens?

For pathogen identification, we're supporting the creation, or generation of a spectra biomarker library. So one thing that we would like to be able to do is obtain as many pathogens as possible, and create tandem mass spectra from their proteins. Then we would put those tandem mass spectra in a database that can be searched against.

We think that this would greatly enable the use of mass spectrometry for pathogen identification.

The flip side of this would be to create DNA sequence databases for all of these pathogens, which would enable you to do PCR, or any other type of sequencing or nucleic acid hybridization type of approach to identify them, but I think that it is much easier to produce a library of tandem mass spectra from proteins from these organisms than it is to sequence all their genomes, and put that into a repository. I think it's much faster, and something that many more people can do.

We are proponents of this type of thing. We have some collaborators at the National Institute of Standards and Technology who are also interested in this type of thing. The NIST is already the agency that maintains these small molecule libraries that the whole world uses to search against to identify their small molecules, so it's really the natural progression for us to work with NIST.

How does your work play into biodefense?

The USDA Cooperative State Research, Education, and Extension Service is giving us funding to use mass spectrometry to identify pathogens. They're interested in and have funded our ideas. That's all part of one of their biodefense programs.

We hope that because mass spectrometry doesn't require these pathogen-specific reagents, it could be used to quickly identify outbreaks of disease, or newly emerging pathogens, or different types of diseases that are popping up in the field that haven't been identified.

I have experience with all this that suggests that mass spectrometry could be faster than some of these routine methods, and it's because the pathogen-derived reagents aren't required.

One thing that really makes all of this work for us is bioinformatics and computing. My laboratory has made significant investments in building bioinformatics resources. Our intention is to distribute these resources to the public. I consider this part of my government service — people are getting something in return for their tax dollars.

There's not a site up now, but we hope to have one up in a few months. The software is in beta testing right now, and in the spring we hope to release it. It's for collating mass spec data, and also performing biomarker searches.

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