To protect crops from diseases and herbivores,

Transcription

1 Plant Defense Systems 37 To protect crops from diseases and herbivores, the United Nations Food and Agricultural Organization estimates that the world s farmers apply over 1.5 million metric tons of the active ingredients found in herbicides, fungicides, and insecticides to their fields each year. When these agents fail, the results can be catastrophic. In 1979 and 1980, a bacterial disease wiped out 60 percent of the rice crop in numerous regions of India; in 1980, a fungus killed the entire wheat crop in many parts of Kazakhstan. Diseases and herbivores constantly threaten crop plants with annihilation. Yet the world is green. This trivial observation is actually interesting. Most plant tissues are not eaten by herbivores or destroyed by bacteria, fungi, or parasitic nematodes (roundworms). Why? Plants cannot run away from these nemeses; instead, they must stand and fight. Wild plant species do this fighting on their own they get no assistance from the chemical agents applied by humans. Understanding how plants defend against disease and herbivory is among the most active research areas in all of plant biology. The experiments we ll review are motivated not only by the excitement of understanding the fundamental biological questions involved, but also by concern about the future of agriculture. Many crops lack desirable disease-fighting traits because such traits have been bred out in an effort to boost productivity. In many cases, massive applications of pesticides are required to make up for the loss of these natural defense systems. How do the molecules involved in plant defense work? Could alleles that are important for defense be safely introduced into crop varieties that are particularly productive and nutritious? To begin answering these questions, let s Diseases and insects cause billions of dollars of crop damage each year. What keeps parasites and herbivores from eating everything? 37.1 Barriers to Entry 37.2 Plant Poisons 37.3 The Cost of Defense 37.4 Responding to Pathogens 37.5 Responding to Herbivores 709

2 710 Unit 7 How Plants Work take a look at defense systems found on the exteriors of plants, and then look at the defenses found inside plants Barriers to Entry The epidermis of a plant is analogous to the skin of an animal. Because it provides a barrier to entry by disease-causing organisms, or pathogens, it serves as an individual s first line of defense. Bacteria, viruses, and fungi must break through the epidermal barrier before they can infect cells, reproduce, and cause disease. Thorns or other structures on the epidermis of a plant are important for thwarting animals that eat plant tissues, or herbivores (plant-eaters). Here we consider two questions about the role of the epidermis in preventing disease and damage. How does the plant epidermis resist entry by pathogens and herbivores? How do pathogens and herbivores overcome these defenses? Cuticle and Cutinase In stems and leaves, the outside surface of epidermal cells is covered with a substance called cuticle (Figure 37.1). Cuticle is made up of a matrix of cross-linked lipid molecules impregnated with the extremely long-chained lipids called waxes. Both elements are hydrophobic and function in limiting water loss from the aerial parts of the plant. The waxy sheet of cuticle also functions in defense, forming a physical barrier that resists penetration by virus particles, bacterial cells, and the spores or growing filaments of fungi. The epidermal cells of roots lack a waxy coating, but have a tough lipid matrix similar to that found in cuticle. Pathogens have several ways of circumventing this barrier. Wounds from herbivores and mechanical damage expose cells to airborne fungal spores, virus particles, and bacterial cells. Bacteria and fungi can also enter stomata when they are open; viruses can be injected directly into plant tissues by aphids, Cross-section of epidermis Cuticle keeps pathogens out Cuticle FIGURE 37.1 Cuticle Coats Epidermal Cells Cuticle forms a barrier that keeps water in and pathogens out. leafhoppers, and other insects that pierce the plant epidermis with their mouthparts to suck phloem sap. Work in Pappachan Kolattukudy s laboratory established that certain fungi have an equally direct way of entering plant tissues: They use enzymes to disrupt the cuticle. Specifically, many pathogenic fungi produce an enzyme called cutinase. Cutinase cleaves the lipid cutin, which forms the cross-linked matrix in cuticle. In this way, fungi hack out a hole in cuticle to expose the cells within. To appreciate the importance of this enzyme, consider work carried out by Linda Rogers and her colleagues. They were able to isolate individuals of the fungus Fusarium solani that contain many copies of the cutinase gene, just one copy, or no functional copies. When Rogers and co-workers infected pea plants with spores from each strain, they obtained the data in Figure The graphs demonstrate a strong correlation between the number of copies of the cutinase gene found in a particular fungal strain and its virulence, or its ability to cause disease. In this case, the ability to break through a plant s first line of defense determines the difference between virulent and benign strains of a pathogen. Strains with more copies of the gene for cutinase are more virulent. Weapons The spines, thorns, and prickles produced by some plants can impede insect and mammal herbivores, just as the cuticle layer helps to thwart viruses, bacteria, and other microscopic pathogens. In some cases, these structures have other functions as well. As Chapter 32 pointed out, the hair-like struc- Percentage of peas infected by fungus Fungus with... many copies of cutinase gene one copy of cutinase gene no copies of cutinase gene Days FIGURE 37.2 Evidence That Cutinase Genes Increase the Virulence of Fungi Cutinase is an enzyme that digests plant cuticle. These data indicate a strong correlation between the number of cutinase genes in Fusarium strains and their ability to infect peas and cause disease. EXERCISE These data show what happened when the researchers applied a high dosage of spores. They repeated the experiment with medium and low doses of spores from each fungal strain. Add lines to the graph predicting the results from medium and low doses.

3 Chapter 37 Plant Defense Systems 711 FIGURE 37.3 Structures That Protect Plants Left: A close-up of a sepal that protects a rose bud. The large bulbous structures projecting from the surface contain chemicals that deter insects and other small herbivores from attacking the young flower. Dozens of simple hairs are visible as well. Right: In adult roses, stems are protected from large herbivores by prickles. QUESTION Why are the leaves of some plants fuzzy? 159 µm tures called trichomes help desert-dwelling plants limit water loss from stomata. As the photographs in Figure 37.3 show, hairs and prickles are weapons analogous to the teeth, pincers, or stingers that animals use to defend themselves from predators. In one case, an army of mercenaries augments these plant weapons. Some Acacia trees native to East Africa have large, bulbous spines that house ant colonies (Figure 37.4a). When a browsing mammal begins to eat leaves or twigs from the tree, the shaking motion stimulates the ants to attack the animal and deliver painful bites. The relationship between the acacias and ants appears to be mutually beneficial, with the trees providing a safe nesting site and the ants providing defense. The ferocity of the ants made P. G. Willmer and G. N. Stone wonder how acacias are able to attract pollinators to their flowers. How could a bee spend enough time on an acacia flower to pick up and deliver pollen if it is quickly attacked by biting ants? To answer this question, the researchers proposed that acacia flowers might produce some sort of chemical deterrent that affects the ants but not bees. After documenting that ants spend much more time at old flowers than at newly opened flowers on the same tree, Willmer and Stone were able to formulate a more specific hypothesis. They proposed that a component of newly opened flowers, such as pollen, contains a chemical that deters ants but not bees. To test this idea, they wiped old flowers with newly opened flowers and documented how long ants stayed during each visit to the treated flowers. FIGURE 37.4 Ant-Guarded Acacias (a) In certain species of Acacia, ants in the genus Crematogaster live in large bulbs at the base of spines. (b) These data show the average amount of time that individual ants spend on acacia flowers of various ages and experimental treatments. QUESTION What would the data in part (b) look like if young flowers did not produce an ant-deterrent compound? (Your answer is the null hypothesis in this experiment.) The results of their experiment are shown in Figure 37.4b. As predicted, old flowers that had been wiped with newly opened flowers were just as unattractive to ants as newly opened flowers. Ant-guarded acacia flowers appear to produce a substance that keeps ants away during the interval when pollination occurs. The study suggests that plants are able to make powerful deterrent compounds, and that in at least some cases these insecticides are species specific. (a) Acacia trees are protected from herbivores by biting ants, which live in large bulbs at the base of the thorns. (b) Does a substance in young flowers keep the ants away from pollinators? Average duration of ant visits (seconds) A substance in young flowers deter ants Young flowers Old flowers Old flowers wiped with young flowers Young flowers wiped with old flowers

4 712 Unit 7 How Plants Work 37.2 Plant Poisons Many plants avoid being eaten by lacing their tissues with poisons. Some of these chemicals are familiar. The flavorful oils in peppermint, lemon, basil, and sage have insect repellent properties. The pitch that oozes from pines and firs contains a molecule called pinene, which is toxic to bark beetles. The pyrethroids produced by Chrysanthemum plants are a common ingredient in commercial insecticides. Molecules called tannins accumulate in the cell vacuoles of many different species. Because they bind to proteins, tannins inactivate digestive enzymes in the herbivores that ingest them. As a result, the herbivores get sick. In humans, small doses of tannins cause a sharp, astringent sensation in the mouth that is prized in apples, blackberries, teas, and red wine. (Tannins also cause structural changes that make proteins less likely to disintegrate; tannins are used to tan animal skins and produce leather.) These plant poisons are similar to cuticle and thorns in an important way: They are always present in the plant. Stated another way, they are produced constitutively which means they do not need a specific stimulus to initiate their production. This is in sharp contrast to the defense systems we ll explore later (in sections 37.4 and 37.5), which are activated only after an infection or attack has begun. Plants that produce poisons constitutively are similar to skunks, stinkbugs, monarch butterflies, and other animals that avoid being eaten by tasting bad or spraying their enemies with noxious chemicals. (Plants that sequester poisons also mount a rapid response to pathogens and herbivores using systems introduced later in the chapter.) Here we ask: Where do plant poisons come from, and how do these molecules act? The Role of Secondary Metabolites In almost every case, plant defense compounds have structures and compositions that are closely related to the molecules required for basic cell activities, such as amino acids. To explain this resemblance, biologists hypothesize that during the evolution of some plant groups, mutations occurred in the genes for enzymes involved in fundamental biosynthetic pathways. The hypothesis claims that these mutations resulted in the production of altered enzymes and the synthesis of new or secondary compounds. If these compounds happened to be toxic to herbivores or pathogens, they would help the mutant individual survive better and reproduce more. Over time, then, the alleles responsible for encoding the required enzymes would increase in frequency in the population. Based on this logic and their chemical similarity to important compounds, plant poisons are referred to as secondary metabolites. To understand how secondary metabolites relate to amino acids and other primary compounds, consider recent work by Monika Frey and colleagues. This research group was interested in a molecule called DIMBOA (2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one) that is found in corn and wheat. DIMBOA is similar in structure to the amino acid tryptophan, but is extremely effective at deterring several of the animal pests that afflict these crops. The steps involved in tryptophan synthesis are well known. Is the synthesis of DIMBOA related to these steps? If so, how? Experiments by other researchers had provided an intriguing hint. When corn plants are fed radioactively labeled molecules that are precursors in the pathway for tryptophan synthesis, the label shows up in DIMBOA as well as in tryptophan. But if the same plants are fed radioactively labeled tryptophan, the label does not show up in DIMBOA. These observations suggest that the synthesis of this secondary metabolite is an offshoot of tryptophan synthesis, as illustrated in Figure 37.5a. To explore this idea, Frey and co-workers analyzed corn plants that have a mutation called Bx1. These individuals cannot synthesize DIMBOA. After an extensive search, the researchers were able to locate the Bx1 gene. Nearby on the chromosome, they found four related loci that they called Bx2, Bx3, Bx4, and Bx5. To determine whether the protein products of these five genes are involved in DIMBOA synthesis, they added the five BX enzymes one by one to cells that were growing in culture and analyzed the products that resulted. Each BX enzyme catalyzed a different reaction, and led to the production of a different intermediate compound in the pathway for DIM- BOA synthesis. The experimental results are consistent with the pathway illustrated in Figure 37.5b. These experiments provide strong support for the hypothesis that secondary metabolites evolved as offshoots of basic synthetic pathways. Now that we know where secondary metabolites come from, let s take a look at how they poison their targets. How Do Caffeine, Nicotine, and Other Alkaloids Act as Poisons? Morphine, cocaine, nicotine, and caffeine are plant secondary metabolites. They are part of a family of chemicals called alkaloids. Members of the alkaloid family are found in about 20 percent of all plant species, and over 12,000 different molecules have been identified in various plant species to date. Although some researchers propose that alkaloids are merely waste products of normal biosynthetic activities, most biologists hypothesize that they are actively synthesized as defense compounds. Michael Wink and associates set up a large-scale study to determine how alkaloids work. They assessed the action of 70 different alkaloids and determined whether each of the molecules has a detrimental effect on DNA structure, DNA synthesis, protein synthesis, membrane permeability, bacterial growth, the survival of insects and worms, or the receptors located in the nerve cells and brains of mammals. The researchers goals were to test whether alkaloids act as poisons and if so, to determine how alkaloids affect herbivores and pathogenic bacteria, viruses, and fungi.

5 Chapter 37 Plant Defense Systems 713 (a) Hypothesis: Secondary metabolites evolved as offshoots of basic synthetic pathways. (b) Experimental support TSB C Tryptophan Amino acid, a primary compound N H N H Tryptophan Enzyme A B TSA NH 2 CO 2 H Precursor compound Precursor compound The enzymes that transform the tryptophan precursor to DIMBOA have been discovered D BX1 E BX2 BX3 BX4 BX5 F Arrows indicate steps in biosynthetic pathway, each catalyzed by a different enzyme DIMBOA Pest deterrent, a secondary metabolite CH 3 O N H N H N H O N H O N OH O N O OH DIMBOA OH O OH O OH O OH O FIGURE 37.5 What Is Secondary About Secondary Metabolites? (a) A secondary metabolite is a molecule that is synthesized as an offshoot of a synthetic pathway for producing a fundamental, or primary, compound. Experimental data suggested that the defense compound DIMBOA is manufactured as an offshoot of tryptophan synthesis. (b) In corn, each of the Bx gene products catalyzes a different reaction involved in the synthesis of DIMBOA. QUESTION Corn seedlings contain over 10 times as much DIMBOA as tryptophan. In this light, are the terms primary and secondary used appropriately? The study results showed that most of the alkaloids tested exhibited strong toxic effects, and that most alkaloids affected more than one aspect of cell biology. An alkaloid that poisons a membrane transport protein might also disrupt DNA structure. Quinine, for example, poisons the enzyme called reverse transcriptase that is found in pathogenic plant viruses; but it also has a strong inhibitory effect on protein synthesis. In interpreting these data, the researchers suggest that natural selection favors the synthesis of alkaloids that poison several target enzymes or structures in the enemy at the same time. The key idea here is that pathogens and herbivores are unlikely to evolve resistance to poisons that affect a large number of important enzymes at the same time. Recall that in Chapter 21, we explored the evolution of resistance in detail. Here, it becomes clear that plant secondary compounds are extremely potent defensive weapons The Cost of Defense If secondary compounds are so effective, why don t all plants produce a lot of them all the time? To address this question, researchers rely on a fundamental observation: Every organism has a finite amount of energy. To understand why this simple fact is important, consider a thought experiment on the weedy mustard plant Arabidopsis thaliana. To begin, suppose that every individual in a population manufactures an average of 100 million ATP molecules in the course of its two-month lifetime. Further, suppose that the individuals in the population vary in how they expend this ATP, and that this variation is based on genetic variation among individuals. For example, some individuals might spend a large percentage of their energy from ATP making defensive compounds and a relatively small percentage making seeds. Others might make few secondary metabolites and invest most of their energy from ATP in reproduction instead. The question is, which type of individual will survive and reproduce best? The short answer to this question is it depends. If pathogens and herbivores are abundant, then most of the poorly defended plants will die. In this case, well-defended plants produce more offspring than lightly defended plants (Figure 37.6a, page 714). As a result, alleles that lead to increased production of secondary metabolites will increase in frequency in the population. But if pathogens and herbivores are rare, then poorly defended plants will produce the most offspring (Figure 37.6b). In this case, alleles that lead to high production of defense compounds will decrease in frequency.

6 714 Unit 7 How Plants Work To summarize these ideas, biologists say that trade-offs occur in the way that individuals allocate their resources. The concept of trade-offs suggests an answer to the question of why all individuals don t produce abundant compounds for their own defense all the time: Manufacturing poisons is energetically expensive. Producing these molecules means that less energy is available for growth and reproduction. Even though these theoretical arguments are logical, it is essential that they be tested rigorously. As an example of experimental studies on the costs of defense, consider work performed by Rodney Mauricio and Mark Rauscher on Arabidopsis thaliana. This species has two major traits that help in its defense against herbivores. Leaves are covered with sharp, hair-like structures called trichomes (look back at Figure 37.3). In addition, both leaves and seeds contain unpalatable molecules called glucosinolates. To explore how these traits affect the ability of A. thaliana to survive and reproduce, Mauricio and Rauscher collected seeds from a large number of individuals growing in a wild population, planted the seeds out in garden plots, and divided each of the plots into two treatments. The researchers regularly sprayed half of the plots with an array of insecticides and fungicides; they did not spray the other half. When the plants were mature, Mauricio and Rauscher collected all of the seeds that had been produced and evaluated the density of trichomes and the concentration of glucosinolates in each individual. What did their data show? Seeds collected from the same parent produced mature individuals with similar levels of trichomes and glucosinolates. Stated another way, related individuals had similar defense traits. This result supports the hypothesis that variation among individuals in these defense characters is at least partly based on variation in their genetic makeup. In the unsprayed plot where herbivores were present, individuals with high concentrations of glucosinolates produced the most offspring. This result is consistent with the prediction that well-defended individuals do better when pest pressure is high. In the sprayed plot with no herbivores, individuals that produced very few hairs and defense compounds produced the most seeds. This result supports the hypothesis that defense is energetically expensive. Stated another way, the ATP and nutrients devoted to synthesis of defensive compounds and structures reduces the number of resources available for seed production. To summarize, the experiment confirmed an important series of predictions about the nature of trade-offs between defense and reproduction. The data also produced a puzzle, however. In both the sprayed and unsprayed treatments, the plants with the fewest trichomes produced the most seeds. Even though trichomes have been shown to be effective against many types of herbivores in many plant species, they appeared to have no effect in the Arabidopsis population that Mauricio and Rauscher studied. If this pattern continued, a population of hairless Arabidopsis would evolve. Alternatively, it is possible that trichomes protect this species against herbivores that did not happen to be present in the year of the study. Further work is needed to distinguish between these two hypotheses. Individuals within populations vary in the concentration of defense compounds High concentration Low concentration (a) Pathogens and herbivores abundant (b) Pathogens and herbivores rare Seeds Seeds FIGURE 37.6 When Do Heavily Defended Individuals Thrive Best? Under what conditions will individuals thrive if they invest a large number of resources in defensive compounds? The diagram illustrates a thought experiment to answer this question. Part (a) shows the results in environments where plants are attacked frequently; part (b) shows the results in environments where plants are attacked infrequently. EXERCISE Make a two-row by two-column table. Label the rows high defense and low defense ; label the columns high pest and low pest. Fill in each box in the table by counting the number of seeds produced by the individuals in this thought experiment. Then answer the question in the figure title.

7 Chapter 37 Plant Defense Systems Responding to Pathogens Manufacturing defensive compounds and structures requires large expenditures of ATP and limits the ability of individuals to grow and reproduce. In light of these findings, it is not surprising that most plants have systems for responding to pathogens and herbivores only after an infection or attack has begun. Response systems are currently the focus of intensive research because of their importance for agriculture. The production of constitutive poisons has been bred out of most crop plants, because toxins taste bad and lower productivity. To fight off diseases and herbivores, then, some crop plants depend solely on their response systems and the application of pesticides. Researchers hope that by gaining a detailed understanding of how response systems work, they will be able to breed or genetically engineer crops that defend themselves more efficiently and are less dependent on pesticides. Section 37.5 explores how plants respond to attacks by insect herbivores; here, we investigate how plants respond to infections by disease-causing viruses, bacteria, fungi, and nematodes. The response systems triggered by these parasites are analogous to the mammalian immune system introduced in Chapter 46. The responses come in two waves: a rapid sequence of events triggered at the point of infection, and a general or systemic reaction occurring throughout the body. The Hypersensitive Response When a virus, bacterium, fungus, or nematode gets inside a plant and begins to grow, the infected cells respond by dying. The rapid and localized death of one or a few infected cells is called the hypersensitive response (HR). If the HR is successful, the pathogen is starved as host cells commit suicide. In several respects, the hypersensitive response in plants is similar to the cell-mediated immune response in mammals, which leads to the death of infected cells. The HR is also extremely effective. Plants that mount a hypersensitive response rarely succumb to disease. How does this response get started, and how is it sustained? What Triggers the Hypersensitive Response? The Genefor-Gene Hypothesis In the early decades of the twentieth century, crop breeders established that plants have disease resistance genes that are inherited according to Mendel s rules. These loci came to be known as resistance (R) genes; many are responsible for triggering the HR. The same researchers also established that the fungi found to cause disease in wheat, flax, barley, and other crops have alleles that cause them to be virulent or avirulent (not virulent) on certain strains or varieties of these crops. The loci associated with virulence or avirulence came to be known as avirulence (avr) genes. In 1956, H. H. Flor published data demonstrating a one-toone correspondence between the resistance alleles found in host plants and the avirulence alleles found in pathogens. Stated another way, Flor showed that certain R alleles and certain avr alleles match. If an R allele in the host matches the avr allele in the pathogen, it means that their protein products must also match in some way. In response to the matching event, an HR occurs. If the R allele in a host and the avr alleles in a pathogen do not match, then no HR occurs and the plant succumbs to disease. The idea that R and avr gene products interact in a specific way came to be called the gene-for-gene hypothesis. What molecular mechanism could be responsible for this pattern? Researchers following up on Flor s work suggested that the HR begins when proteins produced by host plants bind to proteins or other molecules produced by the pathogen. The hypothesis was that R genes produce receptors and avr genes produce ligands meaning molecules that bind to receptors. Figure 37.7 provides a general overview of this model. The first breakthrough in confirming the gene-for-gene hypothesis occurred when researchers from a variety of laboratories around the world were able to clone and sequence a series of R genes from crop plants and avr loci from bacterial and GENE-FOR-GENE HYPOTHESIS Virus Bacterium Fungus R HR avr avr avr R HR R 1. Pathogens (virus, bacterium, or fungus) enter plant cell via wound or connection with infected cell. 2. Proteins and other molecules are released from pathogens. 3. R-gene products bind to certain molecules from pathogens (avr gene products). 4. Binding activates R gene product and triggers protective Hypersensitivity Response (HR). When R and avr gene products do not match, no HR occurs and plant succumbs to disease. FIGURE 37.7 The HR Begins when R Gene Products Bind to AVR Gene Products The gene-for-gene hypothesis predicts that there is a physical interaction between specific R and avr gene products, and that this interaction initiates the hypersensitive response, which protects plants from pathogens. Plant Defenses CD ACTIVITY 37.1

8 716 Unit 7 How Plants Work FIGURE 37.8 Evidence That R and AVR Gene Products Bind to One Another fungal pathogens. These results confirmed that R and avr genes exist and code for products that could interact at the start of an infection. A second major advance, published in 1996, confirmed the gene-for-gene hypothesis by showing that the R and avr products actually do bind to one another. Steven Scofield and coworkers demonstrated this interaction with an R gene in tomato called Pto and an avr gene from Pseudomonas bacteria called avrpto. Figure 37.8 diagrams the researchers experimental strategy. Note that the approach relies on a transcription factor from yeast called GAL4, which was introduced in Chapter 15. In yeast cells, GAL4 activates the transcription of the gene for the -galactosidase enzyme. This enzyme, in turn, can react with a substrate molecule to produce a bright blue product. If the substrate is added to yeast cells in which GAL4 is active, the cells turn bright blue. To confirm that Pto and avrpto actually do bind to one another, the researchers fused the two genes to two different segments of the GAL4 gene. As step 3 in Figure 37.8 shows, Pto was fused to the DNA-binding domain of GAL4, while avrpto was fused to the transcription activation segment of GAL4. GAL4 activates transcription only when the DNA-binding domain and the transcription activation domains are physically attached to one another. When Scofield and co-workers introduced both of the gene constructs into yeast cells that lack GAL4, a complete GAL4 protein was produced, -galactosidase was synthesized, and the cells turned blue (step 6 in Figure 37.8). This result confirmed that R and avr products physically interact, just as predicted by the gene-for-gene hypothesis. Resistance Loci A large number of R loci have now been identified in Arabidopsis, tomato, flax, tobacco, and other plants. Two general patterns are emerging as data on these genes accumulate. First, R genes that are similar in sequence and structure tend to be clustered together on the same chromosome. Second, within a population of plants, there are usually many different alleles at each R locus. (Stated another way, R loci are highly polymorphic.) These observations are important because they provide hints about the history and function of these genes. For example, clusters of similar loci, or what biologists call gene families, are thought to originate through errors in recombination. As Figure 37.9 shows, gene duplication events occur when chromosomes misalign during crossing over. The result of this mutation is a chromosome with an extra copy of a gene. Because the organism with this chromosome already has a functioning copy of the original gene, mutations occurring in the extra copy of the gene do not damage the individual. Instead, the new copy may acquire mutations that make a new function possible. In the case of R loci, the hypothesis is that mutations in the new EVIDENCE SUPPORTING GENE-FOR-GENE HYPOTHESIS Tomato host R (Pto) R protein GAL4 DNA-binding R avr Control cells? Pathogen avr (avrpto) avr protein GAL4 transcription activation β-galactosidase GAL4 DNA-binding GAL4 transcription activation β-galactosidase Yeast cell Experimental cells 1. Question: Do the R and avr proteins interact as the genefor-gene hypothesis predicts? 2. Tool to answer question: The transcription factor GAL4 will only activate transcription when the DNAbinding domain and the transcription activation domains are physically attached. When GAL4 is functioning, it transcribes an enzyme that turns a substrate blue. 3. Construct recombinant genes using R and avr and the two parts of GAL4. 4. Insert recombinant genes into yeast cells to produce two proteins: R + GAL4 part, avr + GAL4 part. 5. Treat transformed cells with substrate to test for presence of functional GAL4. Cells will only turn blue if the two parts of GAL4 are attached. 6. Interpretation: R and avr proteins must have bound to one another. When they did, they created a functional GAL4. Conclusion: The R and avr proteins DO interact as the gene-for-gene hypothesis predicts.

9 Chapter 37 Plant Defense Systems 717 copies gave individuals the ability to recognize and respond to novel avr products and thus new types of pathogens. Why is it significant that many different alleles exist at each R locus? The hypothesis here is that different alleles allow plants to recognize different proteins from the same pathogen. Plants are diploid, so they have two copies of each R gene. If many different alleles exist in a population, then each individual is likely to have two different alleles at each locus. The different alleles allow the host to recognize different avr products. This is important because new avr products constantly arise in pathogen populations via mutation. In combination, then, plants that have different alleles at each of many R loci should be able to recognize and respond to a wide variety of disease-causing agents. The variability observed in R genes supports the hypothesis that plants rely on gene-for-gene interactions to recognize and thwart a diverse variety of invaders. Stated another way, if gene-for-gene interactions underlie some important resistance responses, and if an individual plant has many different R genes, then it can recognize and respond to many different pathogen genes and thus many different pathogens. Before moving on to consider other aspects of the HR, it is interesting to note that mammals have two different sets of genes that are responsible for recognizing pathogens and triggering a R 1 R 2 R 2 R 1 R 1 R 1 R 2 Incorrect synapse of chromatids Bars indicate location of R loci R 2 R 1 R 1 R 2 R 2 One of cross-over products has duplicated R 1 locus FIGURE 37.9 R Gene Families Probably Originated in Gene Duplication Events When biologists find a series of closely related genes clustered together on the same chromosome, as in R loci, they infer that the cluster originated via a series of gene duplication events like the one illustrated here. response. As we ll see in Chapter 46, both types of recognition loci occur in gene families. These loci are found in clusters on the same chromosome, and they are present in high copy number. The genes, which are called the immunoglobulin and MHC loci, are also highly polymorphic. Within any population, many different alleles of each gene exist. The parallels between plant and mammalian disease-response systems are striking. Reactive Oxygen Intermediates (ROI) The interaction between recognition proteins in plants and avirulence molecules from pathogens triggers a series of events. These responses include the production of hydrogen peroxide (H 2 O 2 ), superoxide (O 2 ), and related molecules that are collectively called reactive oxygen intermediates (ROI). These compounds trigger reactions that help reinforce cell walls. In addition, ROI are extremely unstable molecules. As a result, they may also trigger reactions that are responsible for the death of infected cells or that kill the pathogen directly. What steps occur between the R-avr interaction and the end product of the HR cell death? When Massimo Delladonne and co-workers treated plant cells with reactants that lead to the production of ROI, they found that some cell death occurred, but nothing like that induced by the actual R-avr interaction. This observation suggested that the R-avr interaction does not simply lead to the production of compounds required to manufacture ROI. In interpreting their results, the biologists suggested the R-avr interaction must lead to the production of some other molecule that increases or augments the ROI response. Delladonne and associates proposed that the missing molecule might be nitric oxide, or NO. (Nitric oxide is the active ingredient in the anesthetic called laughing gas.) Their hypothesis was inspired by the role that NO plays in the human immune system. The immune-system cells in our bodies frequently use a lethal combination of NO and ROI to kill bacteria and diseased host cells. To test whether the same killing mechanism occurs in plants, the researchers treated Arabidopsis plants with a drug that inhibits the enzyme responsible for producing NO. Then they challenged the plants by spraying the leaves with a bacterial pathogen. Figure 37.10a (page 718) documents the result. Plants that are able to produce normal amounts of NO show the localized cell death typical of the HR. Plants with abnormal NO production are not able to stop the bacterial infection and have large diseased areas in their leaves. The experiment provides strong support for the hypothesis illustrated in Figure 37.10b. Both ROI and NO production appear to be triggered by the R-avr interaction, and both types of molecules seem to be required for the HR. The involvement of NO in the disease response furnishes another parallel between disease-fighting systems in plants and animals. Phytoalexin Production A hallmark of the HR is the production of antibiotic compounds called phytoalexins at the site

10 718 Unit 7 How Plants Work (a) Plants must produce NO to stimulate HR Small lesions produced by HR (b) HYPOTHESIS TO EXPLAIN RESULTS Plant R protein Normal plant Pathogen avr protein Plant treated with drug that inhibits NO production Large diseased areas indicate that HR failed 1. R gene product binds to pathogen molecule. of infection. A phytoalexin is defined as a small (low molecular weight) plant product that is induced by infection and that poisons the disease-causing agent. Because phytoalexins are defined by their function and not their structure, it is not surprising that different plant species produce a diverse array of these molecules. The general idea here is that plant cells not only commit suicide when infected by a pathogen; they produce toxic molecules to poison the pathogen. To get a better appreciation of how phytoalexins work, consider recent research by P. C. Stevenson and colleagues. These researchers were interested in antifungal compounds produced by chickpeas in response to fungi that infect their roots. Chickpeas (also known as garbanzo beans) are an important food in the Middle East and south Asia. However, they are often victimized by a fungal disease called fusarium wilt. Earlier researchers had shown that chickpea roots contain antifungal compounds called maackiain and medicarpin. What Stevenson and co-workers wanted to know is, are these antibiotics effective against fusarium wilt? If so, are they phytoalexins that chickpeas produce in response to infection? To answer the first question, the researchers collected spores from the Fusarium fungus and allowed them to germinate in the presence of various concentrations of maackiain and medicarpin. As the graph in Figure shows, both compounds are effective antifungal agents especially at high concentration. Are these phytoalexins produced in response to infection? When the researchers monitored the concentrations of these antibiotics in chickpea roots over time, they found that strong increases occurred over the span of a week if they inoculated the soil with Fusarium spores. No such increase occurred if the soil was kept free of fungi. This is strong evidence that maackiain and medicarpin are phytoalexins and are part of the response to infection in chickpeas. Even more important, Stevenson and co-workers were able to demonstrate a strong correlation between the ability of chickpeas to ward off fusarium wilt and their ability to produce phytoalexins. Figure shows the results of the experiment. Note that the investigators worked with three varieties of chickpeas and two strains of Fusarium, and that each of the three crop varieties shows a different susceptibility to the Fusarium strains. Some chickpea populations are more resistant to certain fungal strains than others. When the experimenters inoculated individuals from different chickpea varieties with different strains of the fungus and monitored the concentrations of phytoalexins produced, they found that the NO production ROI production Cell death 2. Production of both NO and Reactive Oxygen Intermediates (ROI) is stimulated. 3. Both NO and ROI are required to initiate cell death. FIGURE Evidence That Nitric Oxide Is Involved in the Hypersensitive Response (HR) (a) If Arabidopsis are challenged with a pathogenic bacterium, the HR occurs in normal plants and stops the infection. But if plants are treated with a drug that inhibits nitric oxide (NO) production, the bacterial infection spreads. (b) Researchers suggest that both NO and ROI are required for the cell suicide component of the hypersensitive response. Average germination of fungal spores (%) Both compounds are effective anti-fungal agents, especially at high concentrations Maackiain Medicarpin Concentration (µg ml 1 ) FIGURE Phytoalexins Are Effective Pesticides This graph shows the percentage of fungal spores that germinated when exposed to various concentrations of two different phytoalexins (a type of poison) produced by chickpeas.

11 Chapter 37 Plant Defense Systems 719 ability to produce phytoalexins correlated strongly with the observed level of resistance. Apparently, only certain varieties of chickpeas are able to mount an effective response to certain strains of Fusarium. The data on phytoalexin production in chickpeas are exciting because they suggest a molecular mechanism for the genetic differences in disease resistance among chickpea strains. Time will tell whether this knowledge will lead to the development of new crop varieties with increased disease resistance. Systemic Acquired Resistance (SAR) The HR is fast and leads to localized cell death. Phytoalexin production also occurs at the point of infection. These responses are followed by a slower and more widespread set of events called systemic acquired resistance (SAR). Over the course of several days, SAR primes cells throughout the root or shoot system for assault by a pathogen even in cells that have not been directly exposed to the disease-causing agent. No exposure to fungus (control) Exposure to Fusarium race 1 Exposure to Fusarium race 2 How Do the SAR and HR Interact? Figure illustrates how the HR and SAR are thought to interact. A key point here is that an interaction between R and avr products leads to the production of a signal that initiates SAR. This signal acts globally as well as locally (at the point of infection), and results in the expression of a large suite of genes called the PR (pathogenesis related) loci. The SAR signal qualifies as a hormone because it carries information from one location to another. What is this signal, and what effects does it have? What Molecule Triggers the SAR? When biologists set out to locate the signaling molecule that triggers the SAR, their attention quickly turned to salicylic acid (SA). Salicylic acid is found in a wide variety of plants* and was found to increase dramatically in concentration after tissues are infected with a pathogen. The SA hypothesis became more convincing when researchers in *As an aside, it is interesting to note that SA is very closely related to the active ingredient in aspirin. SA is particularly abundant in willow and aspen trees. Long before medical scientists discovered the pain-relieving qualities of aspirin, Native American people used teas made with willow or aspen bark as a remedy for pain. Mean concentration of phytoalexin (µg g 1 ) Low The ability of chickpeas to produce phytoalexins correlates with the observed level of resistance High R ROI NO Pathogen avr HR SAR signal Other localized defense responses SAR signal 0 Susceptible to races 1 and 2 Resistant to race 1 and susceptible to race 2 Variety of chickpea tested Resistant to races 1 and 2 PR gene transcription Protection of plant from further infection FIGURE Phytoalexins and Disease Resistance To produce the data in these histograms, researchers analyzed production of the phytoalexin medicarpin by three chickpea varieties. One chickpea variety is susceptible to both strains of Fusarium tested, one variety is resistant to one Fusarium strain but susceptible to the other, and one variety is resistant to both Fusarium strains. As the results show, chickpeas that are resistant to certain fungal strains produce phytoalexins only when they are infected with those specific strains. FIGURE The Hypersensitive Response Produces a Signal That Induces Systemic Acquired Resistance This diagram summarizes the current consensus on how the HR and SAR interact.

12 720 Unit 7 How Plants Work (a) EVIDENCE THAT SA ACTS AS A HORMONE (b) EVIDENCE THAT SA DOES NOT INITIATE SAR SA* SA* SAR SA* SA* SA* 1. Inject leaf with radioactively labeled precursor to SA. 2. Infect leaf with virus to trigger SAR. Normal shoot SAR SAR No SA SAR 2. Leaves show normal SAR even though SA could not have originated in root to send message. 3. Later, find labeled SA in uninfected leaves. Grafted root stock that produces enzyme that destroys SA 1. Infect roots with virus to trigger SAR. FIGURE Is Salicylic Acid the Hormone That Triggers Systemic Acquired Resistance? (a) Radioactively labeled SA is transported from the site of an infection throughout the plant. This observation suggests SA is the hormone that initiates SAR. (b) When roots that do not produce SA are grafted onto normal shoot systems and then infected with a virus, the shoots respond with SAR. This observation suggests SA is not the hormone that initiates SAR. several different laboratories showed that applying SA directly to tissues triggers the SAR in a variety of plant species. To confirm SA s role, Thomas Gaffney and co-workers introduced a gene called salicylate hydroxylase into tobacco plants. This gene is not normally found in plants, and it codes for an enzyme that leads to the breakdown of SA. In tobacco plants that received the gene, both SA accumulation and the SAR were abolished. As a result, the transformed plants were susceptible to infection by a wide variety of pathogens. It is still controversial, however, whether SA acts as the SAR hormone or whether it is only a local signal that triggers the expression of genes involved in the SAR. Biologists from several research groups have labeled SA with a radioactive atom and confirmed that it is transported from infected to uninfected tissues. This observation is consistent with the hypothesis that SA actually is a signal that travels throughout the plant (Figure 37.14a). But when investigators in a different laboratory grafted the lower portions of tobacco plants that had been transformed with the salicylate hydroxylase gene onto normal shoots and challenged the roots with a virus, the leaves showed a normal SAR. This observation suggests that SA is not transported throughout the plant body (Figure 37.14b). As this book goes to press, the issue remains unresolved. SA is clearly involved in triggering the SAR. It is still not clear, however, whether SA is a local signal, a global signal, or both. Research on the mechanisms of the hypersensitive response and the systemic acquired response continues. It is clear, however, that these systems are only activated in response to a direct attack by bacteria or viruses and thus minimize the cost of defense. Are analogous systems activated in response to attacks by herbivores? 37.5 Responding to Herbivores Over a million species of insects have already been discovered and named. Most of these species make their living by eating vegetation, seeds, roots, or pollen. How do plants withstand this onslaught of herbivores? In addition to fending off wouldbe predators with thorns and spines and sequestering secondary metabolites that taste bad, plants respond to herbivore attacks once they have begun. When a grasshopper bites a grass leaf, it sets off a series of carefully orchestrated events. Here we explore two of the herbivore response systems that have been researched in some detail. The first response results in the synthesis of insecticides at the point of attack and in nearby leaves; the second leads to the production of compounds that attract enemies of the herbivores. Proteinase Inhibitors In the course of studies to determine why some foods are more palatable and digestible than others, biochemists discovered that many seeds and some storage organs, such as potato tubers, contain proteins called proteinase inhibitors. Proteinase inhibitors block the enzymes found in the mouths and stomachs of animals that are responsible for digesting proteins. When an insect or mammalian herbivore ingests a large dose of a proteinase inhibitor, the herbivore gets sick. As a result, herbivores learn to detect proteinase inhibitors by taste, and

13 Chapter 37 Plant Defense Systems 721 avoid plant tissues containing high concentrations of these molecules. Researchers in Clarence Ryan s laboratory documented that proteinase inhibitors also occur in the leaves of tomatoes and potatoes. During preliminary studies on these compounds, the investigators noticed that the concentration of compounds varied dramatically from plant to plant sometimes by a factor of 10. To explain the variability, the researchers hypothesized that individuals might produce proteinase inhibitors in response to attack by herbivores. They confirmed this idea by documenting that levels of proteinase inhibitors are relatively low in undamaged tomato leaves, but much higher in leaves that were undergoing damage and in leaves at a distance from where damage was occurring. T. R. Green and Ryan followed up on this experiment by allowing herbivorous beetles to attack one leaf on each of several potato plants. In leaves on the same plant that were not attacked, proteinase inhibitor concentrations averaged 336 g per ml of liquid from leaves. In leaves of control plants, where no insect damage had occurred, proteinase inhibitor levels averaged just 103 g per ml of leaf juice. This result supports the idea that a hormone produced at the site of damage travels to undamaged tissues and induces the production of proteinase inhibitors. What is this wound-response hormone? After years of effort, biologists in Ryan s laboratory succeeded in isolating the molecule by purifying the compounds found in tomato leaves and testing them for the ability to induce proteinase inhibitor production. The hormone turned out to be a polypeptide, just 18 amino acids long, called systemin. It was the first peptide hormone ever described in plants. When Gregory Pearce and colleagues labeled copies of systemin with a radioactive carbon atom, injected the hormone into plants, and then monitored its location, they confirmed that systemin moves from damaged sites to undamaged tissues. Currently, work on systemin and proteinase inhibitor production is focused on determining each step in the signal transduction pathway that alerts undamaged cells to danger. As Figure shows, the data indicate that systemin binds to a receptor on the membrane of an undamaged cell. The activated receptor triggers a long series of chemical reactions that eventually result in the synthesis of a molecule called jasmonic acid. Jasmonic acid, in turn, activates the production of at least 15 new gene products, including proteinase inhibitors. In this way, plants build potent concentrations of insecticides in tissues that are in imminent danger of an attack. Recruiting Parasitoids In addition to coping with spines, proteinase inhibitors, and other plant defenses, caterpillars and other herbivorous insects have enemies of their own. In many cases, these enemies are wasps that lay their eggs in the bodies of the herbivores. When a wasp egg hatches inside a caterpillar, the wasp larva begins eating its host from the inside out (Figure 37.16). An organism HOW DOES SYSTEMIN FUNCTION? Damaged cell Undamaged cell Systemin Jasmonic acid Proteinase inhibitors (tastes bad and makes herbivore sick) 1. When a beetle attacks a potato plant, the damaged cells produce the hormone systemin. 2. Systemin binds to receptors on the membranes of undamaged cells. 3. A series of reactions produces jasmonic acid. 4. Jasmonic acid activates transcription of proteinase inhibitors that will deter the herbivore from further attack. FIGURE Signals from Damaged Cells Prepare Other Cells for Attack Systemin is a hormone produced by herbivore-damaged cells that initiates a protective response in undamaged cells. FIGURE Parasitoids Kill Herbivores Parasitoids lay their eggs in caterpillars and other types of herbivores. As the parasitoid larvae grow, they devour the host. This photo shows wasp larvae emerging from a parasitized caterpillar.

14 722 Unit 7 How Plants Work that is free-living as an adult but parasitic as a larva is called a parasitoid. For obvious reasons, parasitoid attacks limit the amount of damage that herbivores do to plants. The observation that parasitoids seem to be common during herbivore outbreaks in croplands prompted a question: Do wounded plants release compounds that actively recruit parasitoids? If so, the distress signal would furnish a novel and effective plant defense system. To explore this idea, T. C. J. Turlings and co-workers collected volatile compounds that were released from corn seedlings during attacks by caterpillars. (A volatile molecule is one that evaporates rapidly and diffuses in the air.) When they analyzed the compounds chemically, the biologists found that insect-damaged leaves produced 11 molecules that were not produced by undamaged leaves. These volatile compounds were not produced by leaves that had been cut with a scissors or crushed with a tool; only insect damage triggered their production. A key question, though, is whether wasps sense the volatile compounds and respond to them. To answer this question, Turlings and colleagues set up the experiment diagrammed in Figure 37.17a. The researchers put corn seedlings on either side of one end of a wind tunnel, put a female wasp at the other end, and recorded which of the two corn plants she visited. As the data at the bottom of the figure show, the wasps were much more likely to visit corn plants that had been damaged by caterpillars than those that had been artificially damaged. The researchers could make artificially damaged plants just as attractive to the wasps, however, by adding caterpillar saliva. H. T. Alborn and colleagues followed up on this result by purifying a large series of substances from the saliva of beet armyworm caterpillars. The researchers were able to show that one of these molecules, called volicitin, induced damaged leaves to emit the volatile compounds attractive to wasps. The message of this work is that plant cells are able to sense a specific molecule that is present in caterpillar saliva. As a result, they are able to recognize when they are being attacked by an herbivore and sound an alarm that recruits help (Figure 37.17b). (a) Wasps can detect herbivore-damaged corn seedlings. Wind tunnel (air blows toward the wasp) Artificially damaged corn seedling Wasp Which side did wasp choose? Number of wasp choices (b) MECHANISM OF COMMUNICATION BETWEEN PLANT AND PARASITOID Volicitin Wind 1. When a caterpillar starts eating a corn leaf, volicitin is released in its saliva.?? Artificial Artificial Caterpillar Caterpillar saliva 2. Volicitin induces plant cells to release volatile molecules. Corn seedling damaged by caterpillar or Artificially damaged corn seedling treated with caterpillar saliva 3. The wasp follows the scent to the source and attacks caterpillar. FIGURE Wasps Hone In on Volatile Compounds That Plants Release in Response to Volicitin (a) In choice tests, wasps are attracted to corn seedlings that have been damaged by caterpillars, or to artificially damaged leaves that have been treated with caterpillar saliva. (b) When plant cells sense volicitin, they release the volatile compounds sensed by parasitoid wasps. QUESTION In part (a), why did the experimenters use artificially damaged leaves as a comparison treatment? Why not use undamaged leaves?