Science news from the Bond LSC

Cut to the signaling chase: EDS1, a plant immune system "central node," is attacked by pathogens

December 8, 2011 | Denise Henderson Vaughn

Two mysteries in the world of plant research are closer to being solved, due to the work of Bond Life Sciences Center investigator Walter Gassmann and members of his lab. Inquiry into the plant immune system led these researchers to confirm that a certain protein operates as a "central node" within the immune signaling system, and in the process they have addressed nagging questions about how a plant communicates internally.

"We've been stymied for years in our field to understand what is the signaling pathway," said Gassmann. He is talking about the "pathway" that plant cells use to "signal" in distress when attacked by a disease organism such as a bacterium or fungus, so that its immune system can activate defenses. This is the first mystery.

Walter Gassmann
Walter Gassmann

In recent decades, scientists have deciphered plants' genetic codes piece-by-piece. Basic biological research on plant immunity has yielded a wealth of understanding as researchers learned how "receptors" within plant cells are able to recognize foreign pathogen proteins and then send out signals that lead to production of defense proteins.

But until now, the exact protein-by-protein chain reaction, the "signaling pathway," has largely eluded scientists. They have persisted in this search because an understanding of how plants naturally protect themselves is among the most critical information sought in ongoing efforts to engineer and breed crops better able to ward off disease. Among others, new soybeans and grape varieties may ultimately result from this research.

This quest has been in the news. Science magazine in July 2011 published a major story about plant immune system proteins and pathogen proteins. Its main hypothesis is that pathogen proteins target "central nodes" in the immune system. Authors had identified a few central nodes, enough cases to form the hypothesis, but they suspected many more were waiting to be discovered.

This brings up the second mystery. Ever since a certain plant gene, "Enhanced Disease Susceptibility1," or EDS1, was discovered a dozen years ago by Jane Parker, currently senior group leader at the Max Planck Institute for Plant Breeding Research in Cologne, Germany, this gene has been studied by many groups and was found in several crop plants. Studies had shown that EDS1 has important regulatory functions in the plant immune system, but exactly what EDS1 does and how it fits into the immune system was unknown.

These two cases of thwarted inquiry are now coming together, as Gassmann's research has simultaneously shed light on both. First, he has established how EDS1 fits into the plant immune system; it is a central node that pathogens are attacking. Second, working in collaboration with Parker and others, he found that messages sent along the "signaling pathway" very likely don't travel at all in the manner expected, which is perhaps why researchers were having a hard time cracking the code.

Science is recognizing these discoveries in papers published in the journal's December 9 issue. In it are two reports from the Gassmann and Parker groups and an accompanying perspective by John McDowell, Virginia Polytechnic Institute.

Gassmann is a professor of plant sciences and a researcher with the Interdisciplinary Plant Group. Coauthors of Gassmann's paper are Saikat Bhattacharjee and Sang Hee Kim, both in MU's Division of Plant Sciences, and Morgan K. Halane, an undergraduate researcher from the Department of English. All are or were members of Gassmann's lab, with Kim currently continuing his post-doctoral studies at Indiana University. The National Science Foundation provided funding for the study.

Arabidopsis plant and tomato bacteria serve as models

Gassmann has conducted his experiments on the plant Arabidopsis. Scientists around the world favor this common weed, related to the mustard plant, as a "research model" because of its relatively simple DNA structure and its quick, six-week life cycle. Arabidopsis was first popularized by plant geneticist George Rédei at MU; now its entire genome has been sequenced and genomic resources and seed stocks are publicly available. Thus, scientists can easily share and compare research results, which builds new knowledge more quickly than if research was conducted on multiple plant species.

Knowledge transfers to many other plants, Gassmann said. "What is true about the immune system of Arabidopis is pretty much also true about the immune system of plants that we care about for our food, fiber and fuel."

Similarly, the bacterium used in the study, Pseudomonas syringae, is a pathogen that causes bacterial speck on tomatoes, but it also infects Arabidopsis, and so it is "a workhorse" for this kind of investigation, Gassmann said. "It's easy to transform; we can put genes that we want into that bacterium. We can compare what happens when the bacterium is recognized by an immune receptor and what happens when it is not recognized."

"Everything we have found with this interaction so far, we also know would be true with a crop plant being attacked by a fungal pathogen. What you find with these model systems you can translate to more important and economical systems," he said. Applying the findings to crops could produce resistant varieties that would reduce the need for fungicides and pesticides.

These two Arabidopsis plants have been genetically modified. The one on the left is modified so that a number of its immune receptors are hyper-activated, which stimulate its immune system to be strongly resistant to pathogens. Such a change encourages the plant to put significant energy into defenses rather than growth, thus stunting it. The plant on the right is genetically identical to the one at left, except that EDS1 has been removed. Without this important gene, the immune system is no longer hyper-activated, and the plant achieves normal growth. This plant would be highly vulnerable to pathogen attack outside of laboratory conditions. The experiment demonstrates the key role that EDS1 plays in the immune system.

Plants vs. pathogens: stealth, recognition, reaction, adaptation

Inside plant tissue, receptors are like scouts, ever on the lookout for invaders. Some are trained to detect "effector proteins," which are substances injected into plants by pathogens. Such proteins are intended to have an effect, that of tricking the plant. Certain plant receptors are capable of intercepting these stealthy tricksters. Most receptors of this type are so specialized that they are equipped to only detect one specific effector protein.

Unlike humans, whose bodies can make antibodies, plants can't adapt their defenses. They have to do battle using whatever tools were within their genetic makeup at germination. So their strategy is to "hedge their bets," said Gassmann, "to have a diversity of immune receptors that hopefully will protect them. Different offspring will have a different variety of these immune receptors to make sure that somebody in the population will survive. But there's nothing that responds within the life of an individual; it can't develop a new immune recognition."

Bacterial pathogens, on the other hand, can evolve quickly. "Because specialized plant immune receptors typically recognize a single protein from a pathogen, and pathogen life cycles are much faster than plant life cycles, the pathogen just evolves. It makes a little change to that effector protein, or deletes it completely, and the plant cannot recognize that pathogen again," Gassmann said.

Because such "co-evolved adapted pathogens" might try something new at any time, plants need additional defenses, beyond relying on receptors that only detect the shapes of specific effector proteins. Scientists have been searching for receptors that can detect other forms of attack, "receptors that the pathogen just can't get around because the receptor is recognizing anything that the pathogen is trying," he said.

Despite a fast life cycle, bacteria function at a certain disadvantage, due to a small genome. "It can't put hundreds of proteins into a plant cell. It only has 20-30 of these (effector) proteins," explained Gassmann. "One prediction, as reported in Science in July, was that these pathogen proteins must be targeting central regulators of the immune system, central nodes. That's the most efficient way to make the plant more susceptible. Like in warfare, it would be advantageous to go for the communications centers, the nodes, the very important centers first."

Gassmann's research on EDS1 supports this hypothesis. He demonstrated that "EDS1 is directly targeted by two bacterial proteins that we know go into the plant cell to muck around in the immune system." This is the first validation that bacteria target EDS1, and the findings help confirm that EDS1 is one of the central nodes sought by scientists. Further, his lab has shown that EDS1 and the receptors associated with it respond to such attacks by activating the immune system.

Surprisingly structured signaling pathway

Scientists already knew that in many types of plant response pathways, signals from receptors are sent by way of a series of signaling proteins, like a wire stretched between a series of telegraph poles. Thus, researchers looking for immune system signals have also focused on finding a step-like course of travel. The signal's ultimate destination is typically the cell nucleus, where the DNA resides that encodes the proteins that the cell needs.

One way scientists have discovered signaling pathways in other response systems has been by blocking the path. "Genetically if you knock out any one of those signaling proteins, you should be able to disrupt the signaling pathway," Gassmann said. Researchers were looking for signaling intermediates along the path. But within the immune system, that approach has been largely unsuccessful. "It's been a real conundrum in the field," he said.

Gassmann found that the step-by-step notion of signal transfer is too simplistic. Instead, the immune system appears to rely on receptors that act as sentinels, and then behave as direct couriers once the danger signal has been detected. "Perhaps that makes sense," Gassmann said. "It is very easy for an enemy to interrupt a telegraph wire by cutting down a pole. It is much harder to intercept multiple messengers."

"Both the immune receptors and the pathogens we have been studying, they're protecting or targeting this one central node (EDS1) itself. That could be the signal, all by itself," Gassmann said. "Rather than passing on a signal to other proteins, it appears that EDS1 and associated receptors themselves move within the cell once the pathogen has been detected," traveling to the cell nucleus to pass on the info needed for defense protein manufacture.

Discoveries open doors for new inquiries

Many scientific discoveries raise new questions just as they answer existing ones. Gassmann's findings are no exception.

Arabidopsis has about 80 effector-triggered immune receptors of the kind the Gassmann lab is studying. The specific pathogen recognized by some of them is known; others are not. "Do all of these (immune receptors) protect EDS1? It seems unlikely, but at this point, that's a question," Gassmann said.

"We've developed a model that now many people can test," he continued. With publication of his findings, Gassmann expects plant researchers will want to investigate whether EDS1 interacts with receptors they may be studying. "It will be interesting to see this expand to more examples."

Perhaps just as crucial is the need for research to "find out why EDS1 is an important protein for the plant immune system as a whole, meaning not just on the level where it is interacting with immune receptors, but its role in cranking up the immune system. We've explained the first half, but not the other half," he said.

"How are the bacterial proteins trying to change EDS1? To neutralize it and make the plant more susceptible? Do they knock it out? That would be my prediction. But we don't know what that change is. I think once we understand all that, we'll be able to get to the applications," Gassmann said.

Applications might protect soybeans and grapes, and eventually help corn and wheat

Pathogens that might eventually be thwarted by applying this new knowledge include Asian soybean rust, grapevine powdery mildew, and a number of downy mildews that attack vegetable crops.

Farmers know that when new varieties of pathogen-resistant crops come onto the market, it is only a few growing seasons before the resistance gene is no longer effective, Gassmann said. To overcome this, "breeders have started pyramiding multiple resistance genes in one plant, so it's very difficult for the pathogen to evolve in one step to evade all of them," he said. But breeding multiple layers of resistance is time-consuming. In his research, he seeks a "deeper understanding of the immune system," which could lead to "a smarter way of breeding or engineering plants, that are more generally resistant to these pathogens."

Arabidopsis, grapevine and soybean all belong to the class of dicots. In the plant kingdom, a deep division separates dicots and monocots. Wheat, maize and rice are monocots. While monocots possess EDS1, they do not have the kind of receptors that were now found to work in tandem to protect EDS1 in Arabidopsis. "Perhaps monocots have a different class of receptors to protect EDS1. But if not, one could imagine being able to engineer an immune receptor that associates with the monocot EDS1 and protects it just like they do in dicots," said Gassmann. "That would be helpful for all the staple crops that we're growing." More research is needed "to gauge whether that's a viable strategy. But ultimately, that's why we're doing this," he said.

Gassmann has this message for farmers. "I don't have a product for you in the next few years, but what we are trying to accomplish is durable field resistance and a reduction in the need for fungicides and pesticides by understanding how the immune system actually works."