The defense strategy of plants
23 October 2009
Plants have developed ingenious strategies for fighting pathogens
Plant Immunity Research Group
RIKEN Plant Science Center
How do plants defend themselves from pathogens such as bacteria or viruses? The Plant Immunity Research Group, led by Group Director Ken Shirasu, is taking on the challenge of unraveling this mechanism. If the mechanism that plants use to defend themselves can be understood, it will be possible to produce plants that are resistant to disease. The Plant Immunity Research Group is also undertaking research on root parasitic plants, which are a big problem in Africa, where they are the main cause of food shortages through attacks on crops such as corn. Unraveling the mechanism of parasitism and creating plants that are resistant to this type of attack would contribute greatly to solving food-shortage problems. “I want to conduct research that will contribute to the global community,” says Shirasu. “I am fascinated by the battle between plants and pathogens, how they attack each other, and how they defend themselves.”
Plants have immune systems too
‘Plant Immunity’, for which Shirasu’s research group is named, may be an unfamiliar term, and little wonder. “I am probably the first person in Japan to have used the term ‘plant immunity’, and it seems that we are still the only researchers who have ever used the term as a name for a laboratory,” says Shirasu. “I’m sometimes asked, ‘You mean plants have immunity too?’ Plants, just like humans, have immune systems because they have to protect themselves from pathogens such as bacteria and viruses. However, plant defense systems differ from ours, which are based on antibodies.”
Mammals have special cells, such as lymphocytes and macrophages, which form the basis of the immune system. These cells circulate on patrol throughout the body, and if they recognize a foreign body (antigen), such as a pathogen that has invaded from outside, they produce antibodies and attack the intruder. Then what about plants? “Plant cells have hard cell walls and are immobile, so there are no immune cells circulating around the plant and recognizing and attacking antigens. Instead, plants have a system that recognizes and attacks pathogens in each individual cell.”
Although the term ‘plant immunity’ has not been used specifically, plant diseases have been studied for many years. The question of how well plants are protected from various pathogens, including microbes such as fungi, bacteria and viruses, has been important ever since plants were first cultivated for human consumption. Pathogen-resistant cultivars were originally produced by crossbreeding of cultivars that were not prone to disease. “Our aim is to shed light on the immunity mechanism that plants use to protect themselves against pathogens. Doing so will lead to the creation of plants that are resistant to diseases.”
Two-phase immune system
Figure 1: The plant immune system.
Phase 1: When a molecule derived from a pathogen (fungus, bacterium, etc.) that has broken the cell wall and invaded the cell is recognized by a MAMPs receptor on the surface of the cell membrane, antimicrobial substances are secreted and attack the pathogen.
Phase 2: Sometimes pathogens (bacteria, etc.) slip through the phase 1 immune system and inject immunosuppressive molecules into the cell, or pathogens (viruses, etc.) invade the cell. In such cases, resistance proteins such as NLR proteins recognize the immunosuppressive molecules, and cell death is triggered. Cells infected by the pathogen die, preventing infection of other cells. Information about pathogen invasion is then transmitted to other cells, which assume a defensive position.
The mechanisms plants use to protect themselves against pathogens are illustrated in Fig. 1. Plant cells have hard cell walls that block invasion by pathogens. However, some pathogens can invade the plant by breaking these cell walls, activating the immune system. Just inside the cell wall is a cell membrane that bears protruding receptors. When a receptor binds to a sugar on part of the flagellum or surface of a bacterium, or to a molecule that does not exist in plants, such as chitin (a component of fungi cell walls), a ‘pathogen invasion’ signal is transmitted throughout the cell. Transmission of this signal causes the production and secretion of antimicrobial substances, which attack the pathogen. This is the ‘phase 1’ immune system. The receptors on the cell membrane, which can recognize many patterns of molecules, are called microbe-associated molecular pattern (MAMP) receptors.
“Plants produce various secondary metabolites. Most of these have the ability to attack pathogens and insects. Furthermore, when one cell recognizes a pathogen, the cell transmits the signal of pathogen invasion to other cells, which prepare for the invasion. However, the pathogens are not often defeated so easily — they slip through the immune system by masking part of their flagella to avoid recognition or by injecting immunosuppressive molecules into the cells to prevent them from producing antimicrobial substances.”
When pathogens inject immunosuppressive molecules into a cell, the ‘phase 2’ immune system is activated. “This system is quite violent and ultimately leads to cell death. Cells infected with the pathogen sacrifice themselves to curb the spread of damage.”
Discovery of essential proteins for recognition of pathogens
So far, the Plant Immunity Research Group has achieved a number of interesting results, including the discovery of three genes that suppress the phase 1 immune system. Shirasu is now focusing on the phase 2 immune system. In particular, he is focusing on immunosensors, which recognize invasion by pathogens, and specifically nucleotide-binding sites and leucine-rich repeat (NLR) proteins. When NLR proteins recognize the invasion of pathogens, cell death is triggered. “We have discovered an essential protein complex for recognition of pathogens by NLR proteins, and we have demonstrated its function.”
Figure 2: Functional analysis of SGT1 protein.
A tobacco leaf with inhibited SGT1 expression is infected with potato virus X bound to a fluorescent protein (GFP). If the SGT1 is overexpressed, virus proliferation can be suppressed (bottom left). However, the virus will proliferate if SGT1 expression is inhibited (top left) or if SGT1 is mutated such that it is unable to bind with HSP90 (right).
This complex consists of three proteins: HSP90, SGT1 and RAR1. Shirasu first showed that the loss of even one of these complex-forming proteins reduces the amount of NLR proteins, making it easier for pathogens to invade. Next, he analyzed the three-dimensional structure of the complex and showed that SGT1 is bound by HSP90 on one side and by RAR1 on the other. Furthermore, when SGT1 was mutated such that it could not bind to HSP90, resistance to pathogens was lost (Fig. 2). He thus showed that the immune system requires binding between SGT1 and HSP90 and that RAR1 strengthens that binding. “We know that this complex also binds to NLR proteins. The shape of these proteins is very important because they act by binding to specific molecules. We think that the loss of this complex may change the shape of the NLR protein so that it ceases to function normally.”
Since the NLR protein is a switch for cell death, its malfunction triggers irreversible consequences. Therefore, NLR proteins are controlled precisely, and if there is even a slight abnormality, or if significant time passes after they are formed, they degrade. “We think this complex plays an important role in the control of NLR proteins,” says Shirasu. “In 2007, a similar complex was discovered in humans and was found to play an important role in the recognition of pathogens. Researchers of mammals, such as humans, are also interested in plant research.”
However, there is a long way to go before we fully understand plant immune systems. “Actually, it seems that the complex that binds to the NLR protein is larger than we thought, and we are only looking at part of it. We need to identify complex-forming proteins one at a time until we can see the whole picture.
“We know from genetic analysis that there are hundreds of types of NLR proteins, yet we still have not succeeded in analyzing the structure of even one. That’s because a stable structure cannot be maintained without the complex, so analysis is impossible. If the structure could be analyzed with the NLR protein and the complex together as a set, it should be possible to figure out what NLR proteins bind to and the mechanism by which they subsequently transmit information to the nucleus and induce cell death.”
In some cases, the NLR proteins bind to the plant’s own proteins instead of molecules originating in the pathogen. “The pathogen attacks by targeting important proteins that are present in the cell. We think that NPR proteins, by binding to proteins that have been attacked and have changed shape, recognize indirectly that a pathogen has invaded. If this method is used, the invasion can be recognized regardless of whether the pathogen is a fungus, a bacterium or a virus.”
Proteins that are involved in plant immune systems in a similar manner to NLR proteins are called ‘resistance proteins’, and their genes are called ‘resistance genes’. Resistance genes are important targets for the creation of cultivars that are resistant to disease, and cultivars in which resistance genes have been introduced by hybridization are already on the market. For example, several resistance genes specific to rice blast fungus have been introduced into the rice cultivar ‘koshihikari BL’. “Introducing resistance genes will surely make cultivars more resistant to pathogens. However, the functions of many genes are still not understood, and the effects of many genes have been reported to disappear after several years. If we can see the whole picture of how NLR proteins recognize pathogens and induce cell death, it could be possible to more efficiently produce cultivars with improved resistance to pathogens.”
For this purpose, Shirasu is trying to understand the big picture of plant immune systems by approaching the problem from a broad perspective and by applying high-level techniques such as proteomic methods, which involve analysis of how changes occur in types and quantities of the proteins made in the cell, and analysis of protein modifications such as ubiquitination or phosphorylation.
Saving Africa from root parasitism
Figure 3: The root parasite Striga.
A parasitic weed of the Scrophulariaceae family that grows in the arid regions of Africa and west Asia. It parasitizes monocots such as rice, corn and sorghum.
The Plant Immunity Research Group also recently started to study root parasitic plants. “I wanted to conduct research that would contribute to the global community,” says Shirasu. “In Africa, damage by root parasitic plants is a very serious problem, and is the main cause of food shortages. The problem has reached the point where it is causing political instability. I would like to present a solution to this problem based on basic research on plant immunity. Root parasitic plants are a mysterious phenomenon in which plants attack plants. There might be a new kind of immune system at work here.” The root parasite that has become the most problematic in Africa is Striga, commonly known as ‘witchweed’ (Fig. 3).
A single Striga plant produces hundreds of thousands of tiny seeds, each about 0.2 mm in length, which are dispersed over large distances due to their light weight. The seeds lie dormant in the ground for many years without budding, through rain and even extended periods of drought. In a Striga attack, the seed buds and affixes to the roots of a host plant when triggered by the presence of certain substances. Striga grows by intercepting moisture and nutrients from its host, causing the host plant to wilt.
Striga is activated by the plant hormones known as ‘strigolactones’, which were discovered about 40 years ago to induce budding of root parasite plants. However, it remained a mystery why plants would purposely excrete substances that would activate potential enemies. Shirasu explains that this puzzle was solved recently when the RIKEN Plant Science Center Cellular Growth and Development Research Team under the leadership of Shinjiro Yamaguchi discovered that strigolactones act to control branching (press release, August 11, 2008). When nutrients such as phosphorus are lacking, strigolactones are secreted to inhibit branching. “Increased branching consumes a lot of nutrients, so this is an energy-saving strategy. Strigolactones inhibit branching and attract mycorrhizal fungi, which parasitize the roots but supply the plant with the phosphorous the fungi produce. Striga exploits this signal that attracts mycorrhizal fungi.” Plants grown in fertile soil do not secrete very much strigolactone, so even when Striga seeds are present, they are rarely activated. In Africa, however, crops are grown in poor, nutrient-deficient soil. Thus, large amounts of strigolactone are secreted by crops and plants, allowing Striga to flourish until the situation becomes unmanageable.
Striga does not parasitize dicots, yet it attacks monocots such as rice and corn. Shirasu is taking note of this difference. “How does Striga distinguish and parasitize its hosts? If we can understand the mechanism of parasitization, we might be able to produce cultivars that won’t be parasitized by Striga.”
Figure 4: Budding and parasitism of Striga.
A Striga seed near the root of a rice plant (a monocot) (top) buds and affixes to the root of the host (A). Near a parasitic plant closely related to Striga (bottom), however, the root of the Striga bud extends toward the plant but then terminates its invasion and retreats (B). Animations are available from the group’s website.
Recently, Shirasu examined the budding of Striga closely, and discovered that Striga also extends its roots toward closely related parasitic plants, but then terminates its invasion and retreats (Fig. 4). Striga does not parasitize its own roots, or the roots of its relatives. “I want to investigate in detail whether Striga terminates its invasion because it recognizes something, or because it is unable to recognize something.”
Developing a strategy
“Plant immunity research fits my personality,” says Shirasu. “Once a famous researcher asked me, ‘What do you find interesting about your research?’ I answered, ‘I am fascinated by the battle between plants and pathogens, how they attack each other, and how they defend themselves.’ He then replied, “You like strategies, don’t you.” I hadn’t noticed this until it was pointed out to me, but it really is true. I was a member of the chess club during my student years, and I enjoy reading war chronicles such as Sanguozhi.”
Even now, an ingenious strategy for understanding plant immune systems is being developed in Shirasu’s mind. “Thinking about strategies for attacking difficult problems is what I enjoy doing most.”