Plant Defenses Against Pathogens PDF

Chapter six HOW PLANTS DEFEND THEMSELVES AGAINST PATHOGENS WHATEVER THE PLANT DEFENSE OR RESISTANCE, IT IS CONTROLLED BY ITS GENES: NON-HOST RESISTANCE – PARTIAL OR HORIZONTAL RESISTANCE – R GENE OR VERTICAL RESISTANCE...

Chapter six HOW PLANTS DEFEND THEMSELVES AGAINST PATHOGENS WHATEVER THE PLANT DEFENSE OR RESISTANCE, IT IS CONTROLLED BY ITS GENES: NON-HOST RESISTANCE – PARTIAL OR HORIZONTAL RESISTANCE – R GENE OR VERTICAL RESISTANCE 208 PREEXISTING STRUCTURAL AND CHEMICAL DEFENSES: PREEXISTING DEFENSE STRUCTURES – PREEXISTING CHEMICAL DEFENSES – INHIBITORS RELEASED BY THE PLANT IN ITS ENVIRONMENT – INHIBITORS PRESENT IN PLANT CELLS BEFORE INFECTION 210 DEFENSE THROUGH LACK OF ESSENTIAL FACTORS: LACK OF RECOGNITION BETWEEN HOST AND PATHOGEN – LACK OF HOST RECEPTORS AND SITES FOR TOXINS – LACK OF ESSENTIAL SUBSTANCES FOR THE PATHOGEN 212 INDUCED STRUCTURAL AND BIOCHEMICAL DEFENSES: RECOGNITION OF THE PATHOGEN BY THE HOST PLANT – TRANSMISSION OF THE ALARM SIGNAL TO HOST DEFENSE PROVIDER – SIGNAL TRANSDUCTION 213 INDUCED STRUCTURAL DEFENSES: CYTOPLASMIC – CELL WALL DEFENSE STRUCTURES – HISTOLOGICAL DEFENSE STRUCTURES: CORK LAYERS – ABSCISSION LAYER – TYLOSES – GUMS NECROTIC STRUCTURAL DEFENSE REACTION: THE HYPERSENSITIVE RESPONSE 214 INDUCED BIOCHEMICAL DEFENSES: INDUCED BIOCHEMICAL NON-HOST RESISTANCE – INDUCED BIOCHEMICAL DEFENSES IN PARTIAL OR HORIZONTAL RESISTANCE: FUNCTION OF GENE PRODUCTS – MECHANISMS OF QUANTITATIVE RESISTANCE – EFFECT OF TEMPERATURE 217 INDUCED BIOCHEMICAL DEFENSES IN THE HYPERSENSITIVE RESPONSE RESISTANCE: THE HYPERSENSITIVE RESPONSE – GENES INDUCED DURING EARLY INFECTION – FUNCTIONAL ANALYSIS OF DEFENSE GENES – CLASSES OF R GENE PROTEINS – RECOGNITION OF PATHOGEN AVR PROTEINS BY THE HOST – HOW DO R AND AVR GENE PRODUCTS ACTIVATE PLANT RESPONSES? – SOME EXAMPLES OF PLANT DEFENSE THROUGH R GENES AND THEIR MATCHING AVR GENES: THE RICE PI-TA GENE – THE TOMATO CF GENES – THE TOMATO BS2 GENE – THE ARABIDOPSIS RPM1 GENE – THE CO-FUNCTION OF TWO OR MORE GENES DEFENSE INVOLVING BACTERIAL TYPE III EFFECTOR PROTEINS – ACTIVE OXYGEN SPECIES, LIPOXYGENASES, AND DISRUPTION OF CELL MEMBRANES – REINFORCEMENT OF HOST CELL WALLS WITH STRENGTHENING MOLECULES – PRODUCTION OF ANTIMICROBIAL SUBSTANCES IN ATTACKED HOST CELLS – PATHOGENESIS-RELATED PROTEINS – DEFENSE THROUGH PRODUCTION OF SECONDARY METABOLITES – PHENOLICS SIMPLE PHENOLIC COMPOUNDS – TOXIC PHENOLICS FROM NONTOXIC PHENOLIC GLYCOSIDES – ROLE OF PHENOL- OXIDIZING ENZYMES IN DISEASE RESISTANCE – PHYTOALEXINS 221 207 208 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S DET\OXIFICATION OF PATHOGEN TOXINS BY PLANTS 236 IMMUNIZATION OF PLANTS AGAINST PATHOGENS: DEFENSE THROUGH PLANTIBODIES – RESISTANCE THROUGH PRIOR EXPOSURE TO MUTANTS OF REDUCED PATHOGENICITY 236 SYSTEMIC ACQUIRED RESISTANCE: INDUCTION OF PLANT DEFENSES BY ARTIFICIAL INOCULATION WITH MICROBES OR BY TREATMENT WITH CHEMICALS 237 DEFENSE THROUGH GENETICALLY ENGINEERING DISEASE-RESISTANT PLANTS: WITH PLANT-DERIVED GENES – WITH PATHOGEN-DERIVED GENES 242 DEFENSE THROUGH RNA SILENCING BY PATHOGEN-DERIVED GENES 244 plant employs against a pathogen or against an abiotic E ach plant species is affected by approximately 100 different kinds of fungi, bacteria, mollicutes, viruses, agent, it is ultimately controlled, directly or indirectly, and nematodes. Frequently, a single plant is attacked by the genetic material (genes) of the host plant and of by hundreds, thousands, and, in leafspot diseases of large the pathogen (Fig. 6-1). trees, probably hundreds of thousands of individuals of a single kind of pathogen. Although such plants may suffer damage to a lesser or greater extent, many survive Nonhost Resistance all these attacks and, not uncommonly, manage to grow well and to produce appreciable yields. A plant may find it easy to defend itself, i.e., to stay In general, plants defend themselves against resistant (immune) when it is brought in contact with a pathogens by a combination of weapons from two arse- pathogenic biotic agent to which the plant is not a host. nals: (1) structural characteristics that act as physical This is known as nonhost resistance and is the most barriers and inhibit the pathogen from gaining entrance common form of resistance (or defense from attack) in and spreading through the plant and (2) biochemical nature. For example, apple trees are not affected by reactions that take place in the cells and tissues of the pathogens of tomato, of wheat, or of citrus trees because plant and produce substances that are either toxic to the genetic makeup of apple is in some way(s) different the pathogen or create conditions that inhibit growth from that of any other kinds of host plants, which, of of the pathogen in the plant. The combinations of course, are attacked by their own pathogens. However, structural characteristics and biochemical reactions apple can be attacked by its own pathogens, which, in employed in the defense of plants are different in dif- turn, do not attack tomato, wheat, citrus, or anything ferent host–pathogen systems. In addition, even within else. Similarly, the fungus that causes powdery mildew the same host and pathogen, the combinations vary with on wheat (Blumeria graminis f. sp. tritici) does not infect the age of the plant, the kind of plant organ and tissue barley and vice versa, the fungus that causes powdery attacked, the nutritional condition of the plant, and the mildew on barley (B. graminis f. sp. hordei) does not weather conditions. infect wheat, and so on. All such unsuccessful plant/ pathogen interactions are thought to represent nonhost resistance. It has been shown recently however, that in WHATEVER THE PLANT DEFENSE at least some related pairings, e.g., the wheat, powdery OR RESISTANCE, IT IS CONTROLLED mildew fungus inoculated on barley, the fungus pro- BY ITS GENES duces haustoria and the host reacts by producing hydro- gen peroxide (H2O2), cell wall appositions under the One concept that must be made clear at the outset is appressoria, and a hypersensitive response in which epi- that whatever the kind of defense or resistance a host dermal cells die rapidly in response to fungal attack.  W H AT E V E R T H E P L A N T D E F E N S E O R R E S I S TA N C E , I T I S C O N T R O L L E D B Y I T S G E N E S 209 100,000 Fungi 500 Bacteria 1,000 Viruses ost resistance. ealthy. 2,500 PHP tative (polygenic) resistance. nfections and symptoms possible. generally survive and produce. 500 Nematodes enic (R gene) resistance. either are resistant and remain y or are susceptible and e severely diseased. FIGURE 6-1 Types of reaction of plants to attacks by various pathogens in relation to the kind of resistance of the plant. Partial, Polygenic, Quantitative, or Horizontal many of those formed in response to attack by a Resistance pathogen or abiotic agent, are important in the defense of most plants against most pathogens. Each plant, of course, is attacked by its own pathogens, When a pathogen attacks a host plant, the genes of the but there is often a big difference in how effectively the pathogen are activated, produce, and release all their plant can defend itself (how resistant the plant is) against weapons of attack (enzymes, toxins, etc.) against the each pathogen. Even when conditions for infection and plants that they try to infect. With the help of different disease development are favorable, a plant, upon infec- combinations of preexisting or induced toxic chemical tion with a particular pathogen, may develop no disease, substances or defense structures, most plants manage to only mild disease, or severe disease, depending on the defend themselves partially or nearly completely. Such specific genetic makeup of the plant and of the pathogen plants show sufficient resistance that allows them to that attacks it. Many genes are involved in keeping a survive the pathogen attacks and to produce a satisfac- plant protected from attack by pathogens. Many of these tory yield. This type of defense or resistance is known as genes provide for the general upkeep and well-being polygenic, general, or quantitative resistance because it functions of plants, but plants also have many genes depends on many genes for the presence or formation of whose main functions seem to be the protection of plants the various defense structures and for preexisting or from pathogens. Some of the latter plant genes code for induced production of many substances toxic to the chemical substances that are toxic to pathogens or neu- pathogen. This type of resistance is present at different tralize the toxins of the pathogens, and these substances levels against different pathogens in absolutely all plants may be present in plants regardless of whether the plant and is also known as partial, quantitative, horizontal, is under attack or not. Plants also have genes that multigenic, field, durable, or minor gene resistance. produce and regulate the formation of structures that Most plants depend on general resistance against their can slow down or stop the advance of a pathogen into pathogens, especially nonobligate parasites, e.g., the the host and cause disease. These structures can also be semibiotrophic or nectrotrophic oomycetes Pythium and present in a plant throughout its life or they may be pro- Phytophthora, the fungi Botrytis, Fusarium, Sclerotinia, duced in response to attack by one of several pathogens and Rhizoctonia, and most bacteria, nematodes, and so or following injury by an abiotic agent. Preexisting on. In at least some polygenic plant–pathogen combina- defense structures or toxic chemical substances, and tions, such as the early blight of tomato caused by the 210 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S necrotrophic fungus Alternaria solani, the more resistant etrate if it is to cause infection. Some structural defenses the varieties are, the higher the constitutive concentra- are present in the plant even before the pathogen comes tion and the more rapid the accumulation in them of in contact with the plant. Such structures include the pathogen-induced pathogenesis related (PR) proteins, amount and quality of wax and cuticle that cover than in susceptible varieties. These PR proteins include the epidermal cells, the structure of the epidermal cell some of the specific antifungal isozymes of chitinase and walls, the size, location, and shapes of stomata and b-1,3-glucanase. Also, total enzyme preparations from lenticels, and the presence of tissues made of thick-walled resistant varieties were able to release elicitors of the cells that hinder the advance of the pathogen on the plant. hypersensitive response (HR) (see later) from purified Waxes on leaf and fruit surfaces form a water- fungal cell walls, whereas enzymes from susceptible vari- repellent surface, thereby preventing the formation of a eties could not. Furthermore, partially purified chitinases film of water on which pathogens might be deposited from tomato leaves could release HR elicitors from ger- and germinate (fungi) or multiply (bacteria). A thick mat minating A. solani spores but not from mature intact cell of hairs on a plant surface may also exert a similar walls. This suggests that, perhaps, constitutively pro- water-repelling effect and may reduce infection. duced hydrolytic enzymes may act as a mechanism of A thick cuticle may increase resistance to infection elicitor release in tomato resistance to the early blight in diseases in which the pathogen enters its host only disease. Quantitative resistance has also been shown to through direct penetration. Cuticle thickness, however, increase in transgenic plants carrying introduced R genes is not always correlated with resistance, and many plant and matching avirulence genes, even though the latter do varieties with cuticles of considerable thickness are not express the hypersensitive cell death. invaded easily by directly penetrating pathogens. The thickness and toughness of the outer wall of epidermal cells are apparently important factors in the Race-Specific, Monogenic, R Gene, or Vertical resistance of some plants to certain pathogens. Thick, Resistance tough walls of epidermal cells make direct penetration by fungal pathogens difficult or impossible. Plants with In many plant–pathogen combinations, especially those such walls are often resistant, although if the pathogen involving biotrophic oomycetes (downy mildews), fungi is introduced beyond the epidermis of the same plants (powdery mildews, rusts), and many other fungi, e.g., by means of a wound, the inner tissues of the plant are Cochliobolus, Magnaporthe, Cladosporium, many bac- invaded easily by the pathogen. teria, nematodes, and viruses, defense (resistance) of a Many pathogenic fungi and bacteria enter plants only host plant against many of its pathogens is through the through stomata. Although the majority of pathogens presence of matching pairs of juxtaposed genes for can force their way through closed stomata, some, like disease in the host plant and the pathogen. The host plant the stem rust of wheat, can enter only when stomata are carries one or few resistance genes (R) per pathogen open. Thus, some wheat varieties, in which the stomata capable of attacking it, while each pathogen carries open late in the day, are resistant because the germ tubes matching genes for avirulence (A) for each of the R genes of spores germinating in the night dew desiccate due to of the host plant. As explained in some detail later, the evaporation of the dew before the stomata begin to avirulence gene of the pathogen serves to trigger the host open. The structure of the stomata, e.g., a very narrow R gene into action. This then sets in motion a series of entrance and broad, elevated guard cells, may also defense reactions that neutralize and eliminate the spe- confer resistance to some varieties against certain of cific pathogen that carries the corresponding (matching) their bacterial pathogens. gene for avirulence (A), while the attacked and a few sur- The cell walls of the tissues being invaded vary in rounding cells die. This type of defense or resistance is thickness and toughness and may sometimes inhibit the known as race-specific, hypersensitive response (HR), advance of the pathogen. The presence, in particular, of major gene, R gene, or vertical resistance. However, some bundles or extended areas of sclerenchyma cells, such as R genes, e.g., Xa21 of rice, do not induce a visible HR. are found in the stems of many cereal crops, may stop the further spread of pathogens such as stem rust fungi. Also, the xylem, bundle sheath, and sclerenchyma cells PREEXISTING STRUCTURAL AND of the leaf veins effectively block the spread of some CHEMICAL DEFENSES fungal, bacterial, and nematode pathogens that cause various “angular” leaf spots because of their spread only Preexisting Defense Structures into areas between, but not across, veins. Xylem vessels seem to be involved more directly in the resistance The first line of defense of a plant against pathogens is and susceptibility to vascular diseases. For example, its surface, which the pathogen must adhere to and pen- xylem vessel diameter and the proportion of large  PREEXISTING STRUCTURAL AND CHEMICAL DEFENSES 211 vessels were strongly correlated with the susceptibility of elm to Dutch elm disease caused by the fungus Ophiostoma novo-ulni. Preexisting Chemical Defenses Although structural characteristics may provide a plant with various degrees of defense against attacking pathogens, it is clear that the resistance of a plant against pathogen attacks depends not so much on its structural barriers as on the substances produced in its FIGURE 6-2 Onion smudge, caused by the fungus Colletotrichum cells before or after infection. This is apparent from the circinans, develops on white onions but not on colored ones, which, fact that a particular pathogen will not infect certain in addition to the red or yellow pigment, also contain the phenolics plant varieties even though no structural barriers of any protocatechuic acid and catechol, both of which are toxic to the fungus. (Photograph courtesy of G. W. Simone.) kind seem to be present or to form in these varieties. Similarly, in resistant varieties, the rate of disease devel- opment soon slows down, and finally, in the absence of structural defenses, the disease is completely checked. and appressorium formation. It was subsequently Moreover, many pathogens that enter nonhost plants shown that ASM accomplished this by increasing the naturally or that are introduced into nonhost plants production and secretion by the plant on the leaf surface artificially, fail to cause infection, although no appar- of coumarins and other toxic phenolics that inhibit ent visible host structures inhibit them from doing so. spore germination and appressorium formation on the These examples suggest that defense mechanisms of a leaf surfaces on which they are present. chemical rather than a structural nature are responsible for the resistance to infection exhibited by plants against Inhibitors Present in Plant Cells before Infection certain pathogens. It is becoming increasingly apparent that some plants are resistant to diseases caused by certain pathogens Inhibitors Released by the Plant in Its Environment because of one or more inhibitory antimicrobial com- Plants exude a variety of substances through the surface pounds, known as phytoanticipins, which are present in of their aboveground parts as well as through the the cell before infection. Several phenolic compounds, surface of their roots. Some of the compounds released tannins, and some fatty acid-like compounds such as by certain kinds of plants, however, seem to have an dienes, which are present in high concentrations in cells inhibitory action against certain pathogens. Fungitoxic of young fruits, leaves, or seeds, have been proposed as exudates on the leaves of some plants, e.g., tomato and responsible for the resistance of young tissues to patho- sugar beet, seem to be present in sufficient concentra- genic microorganisms such as Botrytis. For example, tions to inhibit the germination of spores of fungi Botry- increased 9-hexadecanoic acid in cutin monomers in tis and Cercospora, respectively, that may be present in transgenic tomato plants led to resistance of such plants dew or rain droplets on these leaves. Similarly, in the to powdery mildew because these cutin monomers case of onion smudge, caused by the fungus Col- inhibit the germination of powdery mildew spores. letotrichum circinans, resistant varieties generally have Many such compounds are potent inhibitors of many red scales and contain, in addition to the red pigments, hydrolytic enzymes, including the pectolytic-macerating the phenolic compounds protocatechuic acid and cate- enzymes of plant pathogens. As the young tissues grow chol. In the presence of water drops or soil moisture older, their inhibitor content and their resistance to containing conidia of the onion smudge fungus on the infection decrease steadily. Strawberry leaves naturally surface of red onions, these two fungitoxic substances contain (+)-catechin, which inhibits infection by diffuse into the liquid, inhibit the germination of the Alternaria alternata by blocking the formation of infec- conidia, and cause them to burst, thus protecting the tion hyphae from haustoria although it allows both plant from infection. Both fungitoxic exudates and inhi- spore germination and appressoria formation. Several bition of infection are missing in white-scaled, suscepti- other types of preformed compounds, such as the ble onion varieties (Fig. 6-2). It was noticed that saponins (glycosylated steroidal or triterpenoid com- applications of acibenzolar-S-methyl (ASM) on sun- pounds) tomatine in tomato and avenacin in oats, not flower reduced infection by the rust fungus Puccinia only have antifungal membranolytic activity, they helianthi through the reduction of spore germination actually exclude fungal pathogens that lack enzymes 212 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S (saponinases) that break down the saponin from infect- produce infection substances, such as enzymes, or struc- ing the host. In this way, the presence or absence of tures, such as appressoria, penetration pegs, and haus- saponin in a host and of saponinase in a fungus deter- toria, necessary for the establishment of infection. It is mines the host range of the fungus. not known what types of molecules or structures are In addition to the simple molecule antifungal com- involved in the recognition of plants and pathogens, but pounds listed earlier, several preformed plant proteins it is thought that they probably include various types of have been reported to act as inhibitors of pathogen pro- oligosaccharides and polysaccharides, and proteins or teinases or of hydrolytic enzymes involved in host cell glycoproteins. Also, it is not known to what extent these wall degradation, to inactivate foreign ribosomes, or to recognition phenomena are responsible for the success increase the permeability of the plasma membranes of or failure of initiation of infection in any particular fungi. host–pathogen combination. For example, in a number of plants there is a family of low molecular weight proteins called phytocystatins that inhibit cysteine proteinases carried in the digestive Lack of Host Receptors and Sensitive Sites for system of nematodes and are also secreted by some plant Toxins pathogenic fungi. Constitutively present or transgeni- cally introduced phytocystatins in plants reduce the size In host–pathogen combinations in which the pathogen of nematode females and the number of eggs produced (usually a fungus) produces a host-specific toxin, the by females, thereby providing effective or significant toxin, which is responsible for the symptoms, is thought control of several plants to root knot, cyst, reniform, to attach to and react with specific receptors or sensi- and lesion nematodes. tive sites in the cell. Only plants that have such sensitive Another type of compounds, the lectins, which are receptors or sites become diseased. Plants of other vari- proteins that bind specifically to certain sugars and eties or species that lack such receptors or sites remain occur in large concentrations in many types of seeds, resistant to the toxin and develop no symptoms. cause lysis and growth inhibition of many fungi. However, plant surface cells also contain variable amounts of hydrolytic enzymes, some of which, such as Lack of Essential Substances for the Pathogen glucanases and chitinases, may cause the breakdown of pathogen cell wall components, thereby contributing Species or varieties of plants that for some reason do not to resistance to infection. The importance of either of produce one of the substances essential for the survival these types of inhibitors to disease resistance is not of an obligate parasite, or for development of infection currently known, but some of these substances are by any parasite, would be resistant to the pathogen that known to increase rapidly upon infection and are requires it. Thus, for Rhizoctonia to infect a plant it considered to play an important role in the defense of needs to obtain from the plant a substance necessary for plants to infection. formation of a hyphal cushion from which the fungus sends into the plant its penetration hyphae. In plants in which this substance is apparently lacking, cushions do DEFENSE THROUGH LACK OF ESSENTIAL not form, infection does not occur, and the plants are FACTORS resistant. The fungus does not normally form hyphal cushions in pure cultures but forms them when extracts Lack of Recognition between Host and from a susceptible but not a resistant plant are added to Pathogen the culture. Also, certain mutants of Venturia inaequalis, the cause of apple scab, which had lost the ability to A plant species either is a host for a particular pathogen, synthesize a certain growth factor, also lost the ability e.g., wheat for the wheat stem rust fungus, or it is not to cause infection. When, however, the particular a host for that pathogen, e.g., tomato for wheat stem growth factor is sprayed on the apple leaves during inoc- rust fungus. How does a pathogen recognize that the ulation with the mutant, the mutant not only survives plant with which it comes in contact is a host or but it also causes infection. The advance of the infec- nonhost? Plants of a species or variety may not become tion, though, continues only as long as the growth factor infected by a pathogen if their surface cells lack specific is supplied externally to the mutant. In some host– recognition factors (specific molecules or structures) pathogen combinations, disease develops but the that can be recognized by the pathogen. If the pathogen amount of disease may be reduced by the fact that does not recognize the plant as one of its host plants, certain host substances are present in lower concentra- it may not become attached to the plant or may not tions. For example, bacterial soft rot of potatoes, caused  INDUCED STRUCTURAL AND BIOCHEMICAL DEFENSES 213 by Erwinia carotovora var. atroseptica, is less severe on extracellular microbial enzymes such as proteases and potatoes with low-reducing sugar content than on pota- pectic enzymes. In various host–pathogen combinations, toes high in reducing sugars. certain substances secreted by the pathogen, such as avr gene products, hrp gene products, and suppressor mol- ecules, act as specific pathogen elicitors of recognition INDUCED STRUCTURAL AND BIOCHEMICAL by the specific host plant. In many cases, in which host DEFENSES enzymes break down a portion of the polysaccharides making up the pathogen surface or pathogen enzymes Recognition of the Pathogen by the Host Plant break down a portion of the plant surface polysaccha- rides, the released oligomers or monomers of the poly- Early recognition of the pathogen by the plant is very saccharides act as recognition elicitors for the plant. important if the plant is to mobilize the available bio- chemical and structural defenses to protect itself from Host Plant Receptors the pathogen. The plant apparently begins to receive signal molecules, i.e., molecules that indicate the pres- The location of host receptors that recognize pathogen ence of a pathogen, as soon as the pathogen establishes elicitors is not generally known, but several of those physical contact with the plant (Fig. 6-3). studied appear to exist outside or on the cell membrane, whereas others apparently occur intracellularly. In the powdery mildew of cereals, a soluble carbohydrate that Pathogen Elicitors acts as an elicitor from the wheat powdery mildew Various pathogens, especially fungi and bacteria, release fungus Blumeria graminis f. sp. tritici is recognized by a variety of substances in their immediate environment a broad range of cereals (barley, oat, rye, rice, and that act as nonspecific elicitors of pathogen recognition maize) in which it induces the expression of all defense- by the host. Such nonspecific elicitors include toxins, related genes tested and also induced resistance to sub- glycoproteins, carbohydrates, fatty acids, peptides, and sequent attacks with the fungus. The elicitor alone, in Defense suppression Defense Toxins Enzymes elicitors Polysaccharides Growth regulation Defense reactions Structual Biochemical FIGURE 6-3 Schematic representation of pathogen interactions with host plant cells. Depending on its genetic makeup, the plant cell may react with numerous defenses, which may include cell wall structural defenses (waxes, cutin, suberin, lignin, phenolics, cellulose, callose, cell wall proteins) or biochemical wall, membrane, cytoplasm, and nucleus defense reactions. The latter may involve bursts of oxidative reactions, production of elicitors, hyper- sensitive cell death, ethylene, phytoalexins, pathogenesis-related proteins (hydrolytic enzymes, b-1,3-glucanases, chitinases), inhibitors (thionins, proteinase inhibitors, thaumatin-like proteins), and so on. 214 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S absence of the powdery mildew fungus, did not induce INDUCED STRUCTURAL DEFENSES a hypersensitive response but it did induce an accumu- lation of thaumatin-like proteins in the various cereals. Despite the preformed superficial or internal defense structures of host plants, most pathogens manage to Mobilization of Defenses penetrate their hosts through wounds and natural open- ings and to produce various degrees of infection. Even Once a particular plant molecule recognizes and reacts after the pathogen has penetrated the preformed defense with a molecule (elicitor) derived from a pathogen, it is structures, however, plants usually respond by forming assumed that the plant “recognizes” the pathogen. Fol- one or more types of structures that are more or less lowing such recognition, a series of biochemical reac- successful in defending the plant from further pathogen tions and structural changes are set in motion in the invasion. Some of the defense structures formed involve plant cell(s) in an effort to fend off the pathogen and its the cytoplasm of the cells under attack, and the process enzymes, toxins, etc. How quickly the plant recognizes is called cytoplasmic defense reaction; others involve the the (presence of a) pathogen and how quickly it can send walls of invaded cells and are called cell wall defense out its alarm message(s) and mobilize its defenses deter- structures; and still others involve tissues ahead of the mine whether hardly any infection will take place at all pathogen (deeper into the plant) and are called histo- (as in the hypersensitive response) or how much the logical defense structures. Finally, the death of the pathogen will develop, i.e., how severe the symptoms invaded cell may protect the plant from further invasion. (leaf spots, stem, fruit, or root lesions, etc.) will be, This is called the necrotic or hypersensitive defense reac- before the host defenses finally stop further development tion and is discussed here briefly, with more detailed of the pathogen. treatment a little later. Transmission of the Alarm Signal to Host Cytoplasmic Defense Reaction Defense Providers: Signal Transduction In a few cases of slowly growing, weakly pathogenic Once the pathogen-derived elicitors are recognized by fungi, such as weakly pathogenic Armillaria strains and the host, a series of alarm signals are sent out to host the mycorrhizal fungi, that induce chronic diseases or cell proteins and to nuclear genes, causing them to nearly symbiotic conditions, the plant cell cytoplasm become activated, to produce substances inhibitory to surrounds the clump of hyphae and the plant cell the pathogen, and to mobilize themselves or their prod- nucleus is stretched to the point where it breaks in two. ucts toward the point of cell attack by the pathogen. In some cells, the cytoplasmic reaction is overcome and Some of the alarm substances and signal transductions the protoplast disappears while fungal growth increases. are only intracellular, but in many cases the signal is also In some of the invaded cells, however, the cytoplasm and transmitted to several adjacent cells and, apparently, the nucleus enlarge. The cytoplasm becomes granular and alarm signal is often transmitted systemically to most or dense, and various particles or structures appear in it. all of the plant. Finally, the mycelium of the pathogen disintegrates and The chemical nature of the transmitted signal mole- the invasion stops. cules is not known with certainty in any host–pathogen combination. Several types of molecules have been Cell Wall Defense Structures implicated in intracellular signal transduction. The most common such signal transducers appear to be various Cell wall defense structures involve morphological protein kinases, calcium ions, phosphorylases and phos- changes in the cell wall or changes derived from the cell pholipases, ATPases, hydrogen peroxide (H2O2), ethyl- wall of the cell being invaded by the pathogen. The ene, and others. Systemic signal transduction, which effectiveness of these structures as defense mechanisms leads to systemic acquired resistance, is thought to seems to be rather limited, however. Three main types be carried out by salicylic acid, oligogalacturonides of such structures have been observed in plant diseases. released from plant cell walls, jasmonic acid, systemin, (1) The outer layer of the cell wall of parenchyma cells fatty acids, ethylene, and others. Some natural or syn- coming in contact with incompatible bacteria swells and thetic chemicals, such as salicylic acid and the synthetic produces an amorphous, fibrillar material that sur- dichloroisonicotinic acid, also activate the signaling rounds and traps the bacteria and prevents them from pathway that leads to systemic acquired resistance multiplying. (2) Cell walls thicken in response to several against several diverse types of plant pathogenic viruses, pathogens by producing what appears to be a cellulosic bacteria, and fungi. material. This material, however, is often infused with  INDUCED STRUCTURAL DEFENSES 215 phenolic substances that are cross-linked and further Histological Defense Structures increase its resistance to penetration. (3) Callose papil- lae are deposited on the inner side of cell walls in Formation of Cork Layers response to invasion by fungal pathogens (see Figs. 2- 8C and 2-8D). Papillae seem to be produced by cells Infection by fungi or bacteria, and even by some viruses within minutes after wounding and within 2 to 3 hours and nematodes, frequently induces plants to form after inoculation with microorganisms. Although the several layers of cork cells beyond the point of infection main function of papillae seems to be repair of cellular (Figs. 6-5 and 6-6), apparently as a result of stimulation damage, sometimes, especially if papillae are present of the host cells by substances secreted by the pathogen. before inoculation, they also seem to prevent the The cork layers inhibit further invasion by the pathogen pathogen from subsequently penetrating the cell. In beyond the initial lesion and also block the spread of some cases, hyphal tips of fungi penetrating a cell wall any toxic substances that the pathogen may secrete. and growing into the cell lumen are enveloped by cellu- Furthermore, cork layers stop the flow of nutrients and losic (callose) materials that later become infused with water from the healthy to the infected area and deprive phenolic substances and form a sheath or lignituber the pathogen of nourishment. The dead tissues, includ- around the hypha (Fig. 6-4). ing the pathogen, are thus delimited by the cork layers H CW A S CW AH HC FIGURE 6-4 Formation of a sheath around a hypha (H) penetrating a cell wall (CW). A, appressorium; AH, advancing hypha still enclosed in sheath; HC, hypha in cytoplasm; S, sheath. Epidermis CL Mycelium Starch P grain H Cork I FIGURE 6-5 Formation of a cork layer (CL) between infected (I) FIGURE 6-6 Formation of a cork layer on a potato tuber follow- and healthy (H) areas of leaf. P, phellogen. [After Cunningham (1928). ing infection with Rhizoctonia. [After Ramsey (1917). J. Agric. Res. Phytopathology 18, 717–751.] 9, 421–426.] 216 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S and may remain in place, forming necrotic lesions Formation of Abscission Layers (spots) that are remarkably uniform in size and shape for a particular host–pathogen combination. In some Abscission layers are formed on young, active leaves of host–pathogen combinations the necrotic tissues are stone fruit trees after infection by any of several fungi, pushed outward by the underlying healthy tissues and bacteria, or viruses. An abscission layer consists of a gap form scabs that may be sloughed off, thus removing the formed between two circular layers of leaf cells sur- pathogen from the host completely. In tree cankers, such rounding the locus of infection. Upon infection, the as those caused by the fungus Seiridium cardinale on middle lamella between these two layers of cells is cypress trees, resistant plant clones restrict growth of dissolved throughout the thickness of the leaf, com- the fungus by forming ligno-suberized boundary zones, pletely cutting off the central area of the infection which included four to six layers of cells with suberized from the rest of the leaf (Fig. 6-7). Gradually, this area cell walls. In contrast, susceptible clones have only two shrivels, dies, and sloughs off, carrying with it the to four layers of suberized cells and these are discontin- pathogen. Thus, the plant, by discarding the infected uous, allowing repeated penetration by the fungus past area along with a few yet uninfected cells, protects the the incomplete barrier. rest of the leaf tissue from being invaded by the Abscission layer Healthy area Diseased area Abscission layer FIGURE 6-7 Schematic formation of an abscission layer around a diseased spot of a Prunus leaf. [After Samuel (1927).] (A–C) Leaf spots and shot holes caused by Xanthomonas arboricola pv. pruni bacteria on (A) ornamen- tal cherry leaves; characteristic broad, light green halos form around the infected area before all affected tissue falls off, (B) on peach, and (C) on plum. The shot hole effect is particularly obvious on the plum leaves.  INDUCED BIOCHEMICAL DEFENSES 217 pathogen and from becoming affected by the toxic secre- Necrotic Structural Defense Reaction: Defense tions of the pathogen. through the Hypersensitive Response Formation of Tyloses The hypersensitive response is considered a biochemical rather than a structural defense mechanism but is Tyloses form in xylem vessels of most plants under described here briefly because some of the cellular various conditions of stress and during invasion by most responses that accompany it can be seen with the naked of the xylem-invading pathogens. Tyloses are over- eye or with the microscope. In many host–pathogen growths of the protoplast of adjacent living parenchy- combinations, as soon as the pathogen establishes matous cells, which protrude into xylem vessels through contact with the cell, the nucleus moves toward the pits (Fig. 6-8). Tyloses have cellulosic walls and may, by invading pathogen and soon disintegrates. At the same their size and numbers, clog the vessel completely. In time, brown, resin-like granules form in the cytoplasm, some varieties of plants, tyloses form abundantly and first around the point of penetration of the pathogen quickly ahead of the pathogen, while the pathogen is and then throughout the cytoplasm. As the browning still in the young roots, and block further advance of the discoloration of the plant cell cytoplasm continues and pathogen. The plants of these varieties remain free of death sets in, the invading hypha begins to degenerate and therefore resistant to this pathogen. Varieties in (Fig. 6-9). In most cases the hypha does not grow out which few, if any, tyloses form ahead of the pathogen of such cells, and further invasion is stopped. In bacte- are susceptible to disease. rial infections of leaves, the hypersensitive response results in the destruction of all cellular membranes of Deposition of Gums cells in contact with bacteria, which is followed by desiccation and necrosis of the leaf tissues invaded by Various types of gums are produced by many plants the bacteria. around lesions after infection by pathogens or injury. Although it is not quite clear whether the HR is the Gum secretion is most common in stone fruit trees but cause or the consequence of resistance, this type of occurs in most plants. The defensive role of gums stems necrotic defense is quite common, particularly in dis- from the fact that they are deposited quickly in the inter- eases caused by obligate fungal parasites and by viruses cellular spaces and within the cells surrounding the locus (Fig. 6-10A), bacteria (Fig. 6-10B), and nematodes. of infection, thus forming an impenetrable barrier that Apparently, the necrotic tissue not only isolates the par- completely encloses the pathogen. The pathogen then asite from the living substance on which it depends for becomes isolated, starves, and sooner or later dies. its nutrition and, thereby, results in its starvation and death, but, more importantly, it signifies the concentra- tion of numerous biochemical cell responses and anti- microbial substances that neutralize the pathogen. The XP faster the host cell dies after invasion, the more resist- ant to infection the plant seems to be. Moreover, through the signaling compounds and pathways devel- oped during the hypersensitive response, the latter serves V as the springboard for localized and systemic acquired resistance. PP INDUCED BIOCHEMICAL DEFENSES V A Induced Biochemical Nonhost Resistance T XP As mentioned earlier, nonhost resistance is the resistance that keeps a plant protected from pathogens that are, V through evolution, incompatible with that host. B Although the nature of nonhost resistance is unknown, for a pathogen it can be as big a gap to bridge as the FIGURE 6-8 Development of tyloses in xylem vessels. Longitudi- difference between the features of a potato plant and nal (A) and cross section (B) views of healthy vessels (left) and of vessels with tyloses. Vessels at right are completely clogged with an oak tree, or as close as the difference between the tyloses. PP, perforation plate; V, xylem vessel; XP, xylem parenchyma features of potato and tomato, or barley and wheat. cell; T, tylosis. It appears, however, that in some plant/pathogen 218 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S Z A B C H N H G PS D E F NC FIGURE 6-9 Stages in the development of the necrotic defense reaction in a cell of a very resistant potato variety infected by Phytophthora infestans. N, nucleus; PS, protoplasmic strands; Z, zoospore; H, hypha; G, granular mate- rial; NC, necrotic cell. [After Tomiyama (1956). Ann. Phytopathol. Soc. Jpn. 21, 54–62.] A B FIGURE 6-10 (A) Hypersensitive response (HR) expressed on leaves of a resistant cowpea variety following sap inoculation with a strain of a virus that causes local lesions (in this case, alfalfa mosaic virus). The virus remains local- ized in the lesions. (B) Tobacco leaf showing typical hypersensitive responses (white areas) 24 hours after injection with water (A) or with preparations of bacterial strains B, C, and D. Strain (B), which does not infect tobacco, and (C), which carries a hrp (hypersensitive response and pathogenicity) gene, both induced the hypersensitive response, whereas the third strain (D), a mutant of C that lacked the hrp gene, did not. [From Mukherjee et al. (1997). Mol. Plant-Microbe Interact. 10, 462–471.]  INDUCED BIOCHEMICAL DEFENSES 219 interactions of taxonomically unrelated plants (e.g., compromised in saponin-deficient mutants in which the potato and oak or oak and wheat), nonhost resistance wheat fungus causes a successful infection. This shows is controlled by constitutive defenses and/or defenses that nonhost resistance in some plant/microbe inter- induced by nonspecific stimuli in a nonspecific manner. actions is caused by a direct defense mechanism rather Such defenses include physical topography and the than by recognition events. structures present on the plant, the presence of toxic or In all these examples, the pathogen or the host is the absence of essential compounds, and so on. In other already closely related and nearly fully adopted to the plant/pathogen combinations, in which the plants are characteristics of nonhost resistance presented to it. In taxonomically related (e.g., potato and tomato, barley and less related plants or pathogens, however, in which true wheat), nonhost resistance involves primarily inducible nonhost resistance is found routinely, it is more likely to defenses elicited by the recognition of pathogen-specific be the result of effective nonspecific defenses such as molecules. Some cases of nonhost resistance, however, physical characteristics and nonspecific responses to seem to be controlled by a single gene. wounding and damage done by the pathogen during Some examples of questionable nonhost resistance attempted invasion than to defenses elicited by specific include the resistance of the nonhost pea to the recognition events. There is also, however, the case of Pseudomonas syringae pv. syringae bacterium, which pathogens that have alternate hosts, such as wheat stem infects bean but not pea. The reaction occurs when that rust and barberry and cedar apple rust on apple and bacterium carries a gene that is responsible for elicita- cedar. These are, perhaps, interesting from an evolu- tion of a potentially defensive response in the normally tionary point of view because, presumably, before the nonhost pea, that is expressed as a visible hypersensitive second of the alternate hosts that became a host, it was response. In another example, the potato late blight surely a nonhost. How the rust fungus bridged the two fungus Phytophthora infestans, normally does not infect taxonomically extremely different hosts is not known. the tobacco species Nicotiana benthamiana. The The change in ploidy (from haploid to diploid and back nonhost resistance of the tobacco species, however, is to haploid) was probably involved, but how the fungus lost if the pathogen does not carry an “avirulence-like broke the nonhost resistance of the other host and how gene,” which produces a protein that elicits cell death it used the nonresistant host as a completely coopera- in the tobacco. This is unique in that in other tive host is still a mystery. plant/pathogen combinations, the absence of a single The present consensus is that plants that exhibit “nonhost avirulence gene” does not make the nonhost nonhost resistance against pathogens of other plants do plant susceptible. It would appear, therefore, that if the not need to carry resistance genes that recognize these cell death response to the elicitor controlled by the avir- pathogens because they carry genes that provide the ulence gene really contributes to resistance, then the plants with nonspecific defenses that are fully effective nonhost resistance in such situations is controlled by in protecting the plant from these pathogens. However, more than one component. In still another case, nonhost it may be possible that nonhost resistance, along with resistance in some cereals [wheat to powdery mildew polygenic and monogenic host resistance, forms a strains from another cereal (barley), or in barley to Puc- continuum of resistance that begins to overlap as the cinia rust races from wheat], involves similar gene-for- taxonomic (evolutionary) distance between host and gene interactions and nonhost resistance occurs through nonhost plants becomes closer and results in a complex defense mechanisms involving recognition of an elicitor and continuous network of plant/pathogen interactions. and development of a hypersensitive response. Disease resistance does not always involve pathogen recognition events, but, especially in polygenic or quantitative Induced Biochemical Defenses in Quantitative resistance, it may involve directly various structural or (Partial, Polygenic, General, or Horizontal) chemical defense mechanisms. This also happens in Resistance some cases of nonhost resistance, e.g., in oat roots to the wheat fungus Gaeumannomyces graminis f. sp. In quantitative (partial, polygenic, multigenic, tritici, while they are susceptible to the oat fungus G. general, field, durable, or horizontal) resistance, plants graminis f. sp. avenae. The nonhost resistance of oat depend on the action of numerous genes, expressed con- roots to the wheat fungus is caused by the presence of stitutively or upon attack by a pathogen (induced resist- the saponin compound avenacin in the oat roots, which ance). These genes provide the plants with defensive is toxic to the fungus. This compound is also toxic to structures or toxic substances that slow down or stop the oat fungus, but the latter produces an enzyme that the advance of the pathogen into the host tissues and detoxifies the saponin in oat roots and can infect them. reduce the damage caused by the pathogen. Quantita- The nonhost resistance to the wheat fungus, however, is tive resistance is particularly common in diseases caused 220 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S by nonbiotrophic pathogens. Quantitative resistance more aggressive defense response through the induction may vary considerably, in some cases being specific of cell death and a hypersensitive-like response. The against some of the strains of a pathogen, in others being latter defenses are produced in a manner not unlike that effective against all strains of a pathogen, or providing in a specific host–pathogen interaction, but in the resistance against more than one pathogen. Genes for absence of host R genes. In the quantitatively controlled quantitative resistance are present and provide a basal resistance of the soybean–Phytophthora interaction, level of resistance to all plants against all pathogens soybean tissues actually caused the release of phy- regardless of whether the plant also carries major (or R) toalexin elicitors from the cell walls of the fungus, again genes against a particular pathogen. showing that the plant can play an important role in forcing the release of defense-triggering signals from Function of Gene Products in Quantitative the pathogen. Finally, when five cabbage varieties of different resistance levels were inoculated with a strain Resistance of the cabbage black rot bacterium Xanthomonas Unlike most major (or R) genes involved in monogenic campestris pv. campestris, two varieties were resistant, resistance, which appear to code for components that one was partially resistant, and two were susceptible. In help the host recognize the pathogen and to subse- all varieties there was an increase in the total oxidant quently express the hypersensitive response, genes for activity of peroxidase and superoxide dismutase, accu- quantitative resistance seem to be involved directly in mulation of peroxidases, and lignin deposition. The the expression or production of some sort of structural increases, however, were greater and generally occurred or biochemical defense. Quantitative resistance defenses earlier in resistant than in susceptible varieties. are basically the same ones that follow the hypersensi- However, activity of the antioxidant catalase decreased tive response in monogenic resistance; in quantitative in both resistant and susceptible varieties, but it resistance, however, defenses generally do not follow a decreased more in the resistant variety. The resistant hypersensitive response and cell death because the latter varieties also produced new isozymes of peroxidase and do not usually occur in quantitative resistance. Genes superoxide dismutase that were not produced by the involved in quantitative resistance are present in the susceptible variety. These results suggest that in the same areas of plant chromosomes that contain the genes cabbage–X. campestris pv. campestris system there is involved in defense responses, such as the production of a multilevel resistance similar to a hypersensitive phenylalanine ammonia lyase, hydroxyproline-rich gly- response, although the onset of this response was coproteins, and pathogenesis–related proteins. The delayed when compared to the classical HR. In barley defenses in quantitative resistance, however, develop leaves infected with the fungus Drechslera teres, as slower and perhaps reach a lower level than those in the many as eight pathogenicity-related proteins with race-specific (R gene) resistance. Quantitative resistance thaumatin-like activity were detected. is also affected much more by changes in the environ- ment, mostly of changes in temperature during the Effect of Temperature on Quantitative Resistance various stages of development of resistance. Quantitative resistance is often affected greatly by the temperature in the environment. This effect, however, is Mechanisms of Quantitative Resistance not unique to plants with quantitative resistance, as even Studies of defense mechanisms in diseases with quanti- in plants with monogenic (R) gene resistance, the resist- tative resistance are few and far between. For example, ance of the host may be changed drastically by changes in the early blight of tomato caused by the fungus in temperature. For example, in R resistance-carrying Alternaria solani, all resistant tomato lines had higher wheat, a change in temperature from 18 to 30°C constitutive levels of the pathogenesis-related proteins changes the reaction of wheat plants carrying the Sr6 R chitinase and b-1,3-glucanase than the susceptible lines. gene from rust resistant to rust susceptible. Also, resist- Also, preparations of constitutive enzymes from quanti- ance to rust and powdery mildew was increased in pea tatively resistant, but not from susceptible, tomato and barley, respectively, by low-temperature hardening plants could release elicitors of plant cell death, and pos- of these grain crops. However, a brief “heat shock” may sibly of a hypersensitive response, from the cell walls of cause a brief period of susceptibility of wheat plants to the fungus. These results show that, in this host–plant rust, while it induces resistance to powdery mildew in interaction, the defense responses involve the produc- barley and to cucumber scab, caused by the fungus tion of higher levels of pathogenesis-related proteins in Cladosporium cucumerinum, in cucumber, in which it resistant plants, and the same plants may also induce the also causes an increase in peroxidase activity. There are pathogen to produce elicitor molecules that potentiate a numerous reports of different plants synthesizing a  I N D U C E D B I O C H E M I C A L D E F E N S E S I N T H E H Y P E R S E N S I T I V E R E S P O N S E R E S I S TA N C E 221 variety of pathogenesis-related (PR) proteins in response INDUCED BIOCHEMICAL DEFENSES IN THE to abiotic (low temperature, drought, pollution, wound- HYPERSENSITIVE RESPONSE (RACE-SPECIFIC, ing) as well as to biotic (fungi, bacteria, etc.) stresses. MONOGENIC, R GENE, OR VERTICAL) Some of the PR proteins include PR-1, PR-2 (b-1,3- glucanases), PR-3 (chitinases), and PR-5 (thaumatin-like RESISTANCE proteins), as well as peroxidases. Stressed plants also increase the production of phenylalanine ammonia lyase The Hypersensitive Response (PAL), which is involved in the production of phytoalexins. The hypersensitive response, often referred to as HR, is In a detailed study of the effect of cold hardening of a localized induced cell defense in the host plant at the wheat on its quantitative resistance to infection by the site of infection by a pathogen (Fig. 6-10A). HR is the snow mold fungi, it was found that cold hardening result of quick mobilization of a cascade of defense increases the resistance of wheat to snow mold and also responses by the affected and surrounding cells and the induces changes in the expression (activity) of genes subsequent release of toxic compounds that often kill associated with PR proteins and other defense both the invaded and surrounding cells and, also, the responses, some of them associated with induced sys- pathogen. The hypersensitive response is often thought temic resistance. The most abundant PR proteins pro- to be responsible for limiting the growth of the pathogen duced were chitinase, followed by PAL, b-1,3-glucanase, and, in that way, is capable of providing resistance to PR-1, and peroxidase. Similar PR proteins were pro- the host plant against the pathogen. An effective hyper- duced by plants receiving cold treatment only, but the sensitive response may not always be visible when a level of these proteins was lower and appeared later than plant remains resistant to attack by a pathogen, as it is when the plants were also infected by the snow mold possible for the hypersensitive response to involve only fungi. It is apparent, therefore, that this biotic stress single cells or very few cells and thereby remain unno- induces resistance and that the resistance is further ticed. Under artificial conditions, however, injection of augmented by the fungal infection. This type of resist- several genera of plant pathogenic bacteria into leaf ance has characteristics similar to those of pathogen- tissues of nonhost plants results in the development of and salicylic acid-induced resistance, including the a hypersensitive response. The artificially induced HR expression of PR genes and further enhancement of consists of large leaf sectors becoming water soaked at defense-associated genes following the infection by a first and, subsequently, necrotic and collapsed within 8 pathogen. to 12 hours after inoculation (Fig. 6-10B). The bacteria It should be noted in the aforementioned paragraphs injected in the tissues are trapped in the necrotic lesions that all plants produce PR and other defense-associated and generally are killed rapidly. The HR may occur proteins constitutively and/or following induction by whenever virulent strains of plant pathogenic bacteria biotic and abiotic agents. In some host/pathogen com- are injected into nonhost plants or into resistant vari- binations the level of constitutively produced PR pro- eties and when avirulent strains are injected into sus- teins can be correlated with the level of partial resistance ceptible cultivars. Although not all cases of resistance of the cultivars to the pathogen. There is no proof, are due to the hypersensitive response, HR-induced however, that this correlation is meaningful, especially resistance has been described in numerous diseases since some varieties lack the constitutive production of involving obligate parasites (fungi, viruses, mollicutes, certain PR proteins and yet the plants exhibit partial and nematodes), as well as nonobligate parasites (fungi resistance. It is possible, of course, that plants in the and bacteria). latter varieties have a means of upregulating PR gene The hypersensitive response is the culmination of the expression upon infection that the other varieties lack. plant defense responses initiated by the recognition by As was mentioned already, quantitative resistance the plant of specific pathogen-produced signal mole- depends (a) on the preexisting and induced structural cules, known as elicitors. Recognition of the elicitors by and biochemical defenses provided by dozens and, prob- the host plant activates a cascade of biochemical reac- ably, hundreds of defense-associated genes, (b) on PR tions in the attacked and surrounding plant cells and proteins, which may provide another significant portion leads to new or altered cell functions and to new or of the overall defenses, and (c) on the possible ability of greatly activated defense-related compounds (Fig. 6-11). PR proteins to potentiate a more aggressive response by The most common new cell functions and compounds plant cells to the pathogen invasion by inducing the include a rapid burst of reactive oxygen species, leading pathogen to release molecules eliciting host defenses in to a dramatic increase of oxidative reactions; increased the absence of a gene-for-gene relationship between host ion movement, especially of K+ and H+ through the and pathogen. cell membrane; disruption of membranes and loss of 222 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S Pathogen Pathogen elicitors Pathogen elicitors Host cell receptors Pathogen elicitors Receptors activated Host cell receptors NBS Elicitors react with host cell NBS receptors Defense Receptors become responses activated are activated Nucleus Ovidative burst ROS produced Membranes disrupted Protein binding HR Substances in cell wall cross-linked to DNA alters (localized Lipoxygenases activated jasmonate gene expression response) Phenoloxidases activated and accumulate Phenolics Salicylic acid Programmed cell death Salicylic acid and other Plant signal transducers are produced cell and/or become activated membrane Pathogenesis-related (PR) proteins Plant cell wall Plant cell cytoplasm Systemic Acquired Resistance (SAR) (inhibits intiation of new infections thhroughout the plant) FIGURE 6-11 Diagram of the hypothetical steps in the hypersensitive response defense of plants following inter- action of an elicitor molecule produced by a pathogen avirulence gene with a receptor molecule produced by the match- ing host R gene. cellular compartmentalization (Fig. 6-12); cross-linking kinases); production of antimicrobial substances such of phenolics with cell wall components and strengthen- as phenolics (phytoalexins); and formation of anti- ing of the plant cell wall; transient activation of protein microbial so-called pathogenesis-related proteins such kinases (wounding-induced and salicylic acid-induced as chitinases.  I N D U C E D B I O C H E M I C A L D E F E N S E S I N T H E H Y P E R S E N S I T I V E R E S P O N S E R E S I S TA N C E 223 most other R genes are not known with certainty, but 500 most of them contain domains, such as leucine-rich Conductivity ( mhos) repeats, found in proteins involved in protein–protein 400 interactions. Proteins coded by the tobacco R gene, 300 which protects against tobacco mosaic virus, and the Arabidopsis R gene, which protects against a leaf- 200 spotting bacterium, appear to be present in the plant cell cytoplasm and, therefore, probably recognize pathogen 100 elicitors that reach the cytoplasm. However, the protein encoded by the tomato R gene Cf-9, which provides 0 12 24 36 48 resistance against race 9 of the leaf mold fungus Time (hours) Cladosporium fulvum, and the rice R gene XA21, which provides resistance against many races of the leaf- FIGURE 6-12 Disruption of cell membranes leads to a dramatic increase in cell electrolyte leakage, measured by increased current con- spotting bacterium Xanthomonas oryzae, are trans- ductivity. This occurs when a resistant variety () containing an R membrane receptor-like proteins with a short anchor gene is inoculated with pathogens containing an avirulence gene cor- and a protein kinase, respectively. The last two R gene responding to the R gene. Same variety inoculated with a pathogen products, therefore, apparently recognize pathogen- lacking the avirulence gene (); another variety, susceptible to both produced molecules as they approach or come in contact pathogens (, ). [From Whalen et al. (1993). Mol. Plant-Microbe Interact. 6, 616–627.] with the plant cell membrane. Genes Induced during Early Infection The hypersensitive response occurs only in specific Through recent methodology [suppression subtractive host–pathogen combinations in which the host and the hybridization (SSH), cDNA library construction, pathogen are incompatible, i.e., the pathogen fails to expressed sequence tag (EST) determination, large-scale infect the host. It is thought that this happens because DNA sequencing, and DNA microarrays], it is now pos- of the presence in the plant of a resistance gene (R), sible to detect and identify numerous plant genes (or which recognizes and is triggered into action by the elic- ESTs) and their organization, including those induced itor molecule released by the pathogen. The pathogen- during compatible or incompatible interactions between produced elicitor is, presumably, the product of a plant pathogens and their hosts. DNA microarrays, pathogen gene, which, because it triggers the develop- especially, can provide extremely useful information on ment of resistance in the host that makes this pathogen the expression patterns of thousands of genes in paral- avirulent, is called an avirulence gene. For several lel. Earlier studies, for example, of a compatible inter- pathogens, primarily bacteria, avirulence genes have action of Phytophthora infestans and potato, 43 genes been isolated and the proteins coded by them have been appeared to be induced, 10 of which showed increased identified. The first avirulence gene product to be iden- activity as a result of the infection. Some of them were tified was the protein of the avirulence gene D (arvD) of homologous to genes already known to be activated the bacterium Pseudomonas syringae pv. glycinea. This during infection, e.g., for b-1,3-glucanase, some have was shown to be an enzyme involved in the synthesis of homology to enzymes involved in detoxification, and substances known as syringolides. The latter have the some code for proteins that had not been reported ability to elicit the hypersensitive response in soybean earlier to be induced by infection. When genes expressed varieties that carry the resistance gene D complementary by rice seedlings 48 hours after inoculation with the to avrD of the bacterium. fungus Magnaporthe grisea were examined, of 619 ran- More than 20 resistance (R) genes have been isolated domly selected clones, 359 expressed sequence tags that from a variety of plants such as corn, tomato, tobacco, had not been described before. When 124 of 260 ESTs rice, flax, and Arabidopsis, a model plant used for that showed moderate and high similarity were organ- experimental purposes. The corn R gene Hm1 for north- ized according to their suspected function, the largest ern leaf spot codes for an enzyme that inactivates the group (21%) contained (24) stress or defense response HC toxin of the fungus Cochliobolus carbonum, the genes. When looked at from a different angle, many of cause of northern leaf spot of corn, whereas the tomato the genes were new and not described previously, but gene Pto, that confers resistance to the tomato speck- several had been described before and were known to causing bacterium Pseudomonas syringae pv. tomato, be involved in the infection process; one, for example, codes for a protein kinase enzyme that most likely plays being the rice peroxidase gene, which is expressed a role in signal transduction by triggering other enzymes during the infection of rice with the bacterial blight into action. The functions of the proteins encoded by pathogen Xanthomonas oryzae pv. oryzae. 224 6. H OW P L A N T S D E F E N D T H E M S E LV E S AG A I N S T PAT H O G E N S In more recent studies, almost 2,400 genes of Ara- the promoter regions of defense-related genes is also bidopsis were examined for transcriptional changes that critical for understanding how defense gene expression may occur after inoculation with the incompatible is regulated. It is now possible to identify novel regula- fungal pathogen Alternaria brassicicola or after treat- tory elements in the promoter regions of coregulated ment with defense signaling compounds such as salicylic genes with bioinformatics tools. Genes that participate acid (SA), methyl jasmonate (MJ), or ethylene. More in the same biochemical, cellular, or developmental than 700 of the genes exhibited transcriptional changes processes may be controlled by the same sets of tran- in response to one or more of the treatments. Based on scription factors and, therefore, promoter sequences of similarity of the sequences of these genes to known gene such genes may also have some common regulatory sequences, the majority of the activated genes were sequences. already known, but an additional 106 genes were also activated. Treatments with salicylic acid and methyl jas- Classes of R Gene Proteins monate activated 192 and 221 genes, respectively, but they also repressed the transcription of 131 and 96 The various plant R genes, regardless of the type of genes, respectively. Of the identified genes that were pathogen (bacterial, fungal, or viral) to which they activated, a number of them are involved in the oxida- confer resistance, have many structural similarities. It tive burst, in antimicrobial defense, cell wall modifica- appears that most, if not all, R genes exist as clustered tion, phytoalexin production, and defense signal gene families. So far, depending on structure and func- transduction. There appears to be a high level of inter- tion, R genes can be subdivided into five classes (Fig. 4- action among signaling pathways regulated by pathogen 14, Table 4-5) (The R-like gene Hm1, which encodes a infection or by treatment with SA, MJ, or ethylene. For detoxifying enzyme, does not fit and does not follow the example, of 2,375 ESTs analyzed simultaneously, 169 gene-for-gene concept.) (1) R genes, like Pto, encode a were regulated by more than one pathway. Of these, 55 serine–threonine protein kinase that plays a role in genes were coinduced and 28 genes were corepressed by signal transduction. (2) R genes, like Xa21 of rice, which SA and MJ in local tissue, but only 6 genes were co- encode a transmembrane protein rich in extracellular induced in both local and systemic tissue. leucine repeats and a cytoplasmic serine–threonine kinase, function as receptors of kinase-like proteins and transmit the signal to phosphokinases for further ampli- Functional Analysis of Plant Defense Genes fication. (3) R genes, like the tobacco N1 gene, the flax Expression of dozens or hundreds of genes at a partic- L6 gene, and the RPP5 Arabidopsis gene, encode pro- ular physiological state, such as at a certain time inter- teins that are cytoplasmic. These cytoplasmic proteins, val after inoculation with a pathogen or a related in addition to leucine-rich repeats, also have a site that treatment, implies the involvement of these genes in that binds to nucleotides (NBS) and a domain (TIR) with physiological state. Determination, however, of which significant homology to the Toll/interleukin 1 receptor; specific gene is responsible for a certain function such proteins may serve as receptors that activate the requires that the study of the function of each gene be translocation of a transcription factor from the cyto- carried out individually. This is a very difficult task, plasm to the nucleus where it activates transcription of partly because of the large number of genes contribut- the genes related to hypersensitive response. (4) Another ing to the same function and because many of the same group of cytoplasmic R proteins also have LRR and functions are carried out by several different genes. Also, NBS, but have a coiled coil domain that contains a several plant gene families consist of 100 or more putative leucine zipper domain, such as in RPS2 and members, and in some gene families related to tran- RPM1. (5) R genes, like the tomato Cf2–Cf9 genes, scription factors, most of the genes are particularly asso- encode proteins that consist primarily of leucine-rich ciated with defense responses. Nevertheless, candidate repeats and are located outside the cell membrane but genes identified in microarray experiments can be sub- are attached to the membrane with a transmembrane jected to detailed functional analysis in planta through anchor. Such R gene-coded proteins may serve as recep- several strategies, including posttranscriptional silenc- tors for the extracellular or intracellular elicitor mole- ing, overexpression of genes, gene knockout experi- cules produced as the result of expression of the ments using insertional mutagenesis via transposon or corresponding avr gene. For example, in the case of T-DNA, through promoter trap strategies, and others. avr9, the elicitor molecule is a peptide consisting of 22 The generation of transgenic plants for the functional amino acids and binds to the receptor product of the analysis of genes is both time-consuming and may show Cf9 R gene. A potential sixth class of R proteins may high variation of transgene expression. The identifica- be coded by Arabidopsis genes RPW8.1 and RPW8.2, tion of transcription factors and their binding sites in which individually provide resistance against a broad  I N D U C E D B I O C H E M I C A L D E F E N S E S I N T H E H Y P E R S E N S I T I V E R E S P O N S E R E S I S TA N C E 225 range of powdery mildew pathogens. RPW8 proteins