Plant Defences Against Fungal Attack: Biochemistry

Deanna L Funnell, University of Kentucky, Lexington, Kentucky, USA Christopher L Schardl, University of Kentucky, Lexington, Kentucky, USA
A wide variety of defence molecules are produced by plants either before fungal attack (preformed) or in response to an attack (induced).

Secondary article
Article Contents
. Introduction . Basic Concepts . The Fungus Attacks . Preformed Plant Defences . Induced Plant Defences . Conclusions

Introduction
Fungi and fungus-like microorganisms are extremely important agents of diseases that limit the productivity and kill o large numbers of plants. Their devastating eects range from economic hardship to poisoning of food supplies (such as ergotism) and horrendous famines such as the Irish potato famine that lasted from 1845 to 1852. Even under natural (nonagricultural) conditions fungal diseases of plants are apparent. Hundreds of millions of years during which plants and their pathogens coevolved have resulted in numerous and sophisticated defences, as well as many ways that fungi can thwart defences to make their living. Fortunately, most fungi fail to infect most plants, and the small number that infect a particular plant species are incapable of killing o all individuals of that species. If the plants could not stay one step ahead of their pathogens they would have become extinct and taken most life forms with them. Therefore, studying mechanisms of plant defences is of great importance to human well-being, as well as great academic interest in evolutionary biology.

Plant defences can be classied as constitutive (preformed) or as induced in response to fungal attack. However, at least some constitutive defences can be induced at a higher level or become more active following induction, and some inducible defences have low constitutive levels.

The Fungus Attacks

Fungal pathogens that live in the soil and infect roots face dierent problems from those infecting above-ground parts (leaves, stems, owers, fruit). Many root-infecting fungi produce spores that can be viable for years and highly tolerant of desiccation, heat and other environmental factors. Some oomycetes, such as Pythium and Phytophthora species, produce agellated, motile zoospores that respond to root exudates and swim toward sites suitable for successful attachment and germination. Nonmotile spores germinate specically in the presence of host root exudates. Fungi that infect above-ground parts release numerous spores that become airborne or spread by rain splash. Appropriate growth, dierentiation and interaction with the leaf surface is essential for these fungi to infect plants. For example, once a rust fungus spore lands on a leaf it germinates and the growing germ tube tracks the contours of the leaf surface to a stomate, its point of entry into the plant. Other fungi enter the leaf epidermis directly by means of enzymes, physical force or both. Most fungi produce enzymes that may be involved in attachment, penetration, and ramication of plant tissues. These enzymes are a two-edged sword for the pathogen because they release fragments of cell walls (soluble oligosaccharides) that may be toxic to plant cells or may cause the plant to launch a defensive response.

Basic Concepts
Plant disease is a signicant alteration in normal physiological or biochemical development of the plant. Disease agents can be abiotic or biotic. This article considers fungi and fungus-like protists (oomycetes and myxomycetes) as biotic disease agents, though many of the principles raised here also apply to plant interactions with viruses, bacteria, nematodes and parasitic plants. Any given plant species is resistant to infection by all strains of most fungal species. This level of resistance is termed nonhost resistance. A pathogen is dened as the causal agent of disease on at least some members of a plant species, the host species. However, individual strains of a given pathogen (designated as races or pathovars) may not be able to infect all members or varieties (designated as genotypes, cultivars or breeding lines) of a plant species. Resistance exhibited by those members of a plant species refractory to infection by a fungal race or pathovar is called cultivar resistance.

Preformed Plant Defences

Physical barriers or plant surfaces, though not extensively studied, probably play major roles in defence. Thickened cuticle layers and secretions from trichomes (leaf glands)
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Plant Defences Against Fungal Attack: Biochemistry

may signicantly inhibit fungal penetration. A variety of preformed antibiotics, collectively called phytoanticipins, are stored in plant cells. A classic example is protocatechuic acid, a phenolic phytoanticipin that gives red onions their colour and protects against the fungus, Colletotrichum circinans. Another group of phytoanticipins are the saponins steroids and terpenoids linked to sugar moieties which create pores in membranes. In certain plants, saponins have a role in nonhost resistance; pathogens are resistant to the particular saponins of their hosts whereas nonpathogens are highly sensitive (Sandrock and VanEtten, 1998).

Induced Plant Defences

Formation of papillae
In an early stage of defence, particularly against a nonpathogen, the plant often deposits a large amount of cell wall material at the site of attempted fungal penetration. The resulting plug, which also contains high levels of antimicrobial proteins, is called a papilla. Rapid formation of papillae may prevent penetration and intracellular growth of the fungus, or delay its invasion long enough for other defences to become eective.

Within minutes of fungal penetration, changes occur in plant membrane integrity and ion uxes, resulting in a large inux of protons and eux of potassium ions. Additionally, the plant generates toxic active oxygen species such as superoxide anion (5 ) and hydrogen peroxide (H2O2). The role of H2O2 is multifarious. In addition to being antibiotic, H2O2 also crosslinks host cell wall components to strengthen the barrier against fungal penetration; furthermore, H2O2 acts as a second messenger to turn on defence-related genes. Then, genes are induced for secondary metabolism, generating more cell wall polymers (especially lignin and suberin) and production of antimicrobial compounds (phytoalexins) (Figure 1). Other cell wall modications occur, such as papilla formation and increased deposition of hydroxyprolinerich glycoproteins that are also involved in H2O2 crosslinking and further restrict fungal penetration and growth. Ultimately plant cells die, evidently as a programmed response to pathogen activity.

Cell wall-degrading enzymes

Proteins that are produced in response to infection and may be directly involved in defence are known as pathogenesis-related proteins (PR-proteins). Some of these proteins are enzymes that hydrolyse and break up biological polymers. Examples are chitinases (PR-3) and b1,3-glucanases (PR-2), which degrade fungal and oomycete cell walls. Both activities have been shown to inhibit fungal growth in vitro. Genes for these enzymes have been constitutively expressed in transgenic plants, resulting in delayed fungal growth following infection. PR-2s and other b-1,3-glucanases are also involved in growth and development in healthy plants.

The hypersensitive response (HR)

HR may occur as a result of attempted infection by a nonpathogen or by a pathogen of a host with cultivar resistance. The response is rapid death of a small number of plant cells at the site of infection, restricting nutrient uptake by the fungus and presumably preventing further fungal growth (Figure 1). Antimicrobial substances released by dying or dead plant cells can kill some (but not all) invading pathogens. HR can be induced by elicitors produced by the fungus (exogenous elicitors) or the host (endogenous elicitors). Exogenous elicitors may be fungal cell wall fragments or products of fungal genes. In the latter case, such elicitors are produced by a pathogen and are involved in initiating a resistance response in one or more cultivars of the host species. Fungal cell wall fragments also elicit responses, but are not cultivar- or species-specic. Endogenous elicitors are usually plant cell wall degradation products, and are also nonspecic. In some systems these nonspecic elicitors may be critical players in defence, but the fungus produces specic suppressors of host defence responses. Elicitors trigger a cascade of signal transduction in the plant. Rapid changes in protein phosphorylation is evident, indicating kinase and phosphorylase activities typical of signal transduction. Likewise, roles for G proteins and phospholipase C are indicated.
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Antimicrobial proteins
Two other PR-proteins have demonstrated toxicity to fungi. PR-1s are produced by numerous plant species in response to infection, and help protect against oomycetes. PR-5s isolated from numerous plants exhibit antifungal activity and, by structural analysis, some may have protease or protease inhibitor activity (Skern et al., 1990). This group also includes osmotins, so named because they are also induced during water decit or salt stress. Unlike other defence proteins, which are nonspecic, osmotins appear to act against specic pathogens, perhaps via membrane permeabilization. In the case of the oomycete Phytophthora infestans, a tomato osmotin caused hyphal-tip lysis as well as growth inhibition (Woloshuk et al., 1991). Thionins are small, basic, cysteine-rich polypeptides with antimicrobial activity. They are toxic to bacteria, plants and animals, as well as to fungi. Their mode of action appears to be by membrane permeabilization and

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Plant Defences Against Fungal Attack: Biochemistry

Figure 1 Hypersensitive response: early plant responses to fungal invasion include changes in cell wall structure. Cytoskeletal rearrangements may be involved in deposition of cell wall materials. Defence genes are activated, including those that result in the production of phytoalexins and degradative enzymes. The host cell undergoes programmed cell death. Hydrolytic enzymes such as b-1,3-glucanases (PR-2s) and chitinases (PR-3s) are released into the plant intercellular spaces and can degrade fungal cell walls. Signal molecules are also released that trigger defence gene expression in adjacent cells. Redrawn from the work of M. Hazzard with permission from Medical Arts & Photography, Chandler Medical Center, University of Kentucky (1999).

involvement in redox reactions. A thionin gene was constitutively expressed in an Arabidopsis thaliana ecotype susceptible to Fusarium oxysporum, and the transgenic plants were resistant to the fungus (Epple et al., 1997), thus indicating a direct role of thionins in defence.

Tolerance to fungal toxins

Some pathogenic fungi produce toxic compounds that may be involved in disease development on their plant hosts. In some cases, the isolated toxin alone can produce symptoms similar to infection by the fungus. It is therefore surprising that only a single example is known of disease resistance conferred by a plant detoxication mechanism. This is a reductase encoded by the maize gene Hm1, responsible for eliminating the activity of the HC-toxin produced by Cochliobolus carbonum. It is possible that many analogous examples remain to be elucidated. Alternatively, perhaps such mechanisms are rarely needed since plants have other strategies to respond rapidly to and kill the invading pathogen. Such strategies as rapid host cell death (HR), erection of infection barriers (lignins, suberins, etc.), and production of antibiotic metabolites and proteins may simply be more generally eective at stopping pathogens.

mechanisms of defence against some pathogens are ineective or may actually help others. Preliminary genetic and molecular genetic studies of plantpathogen interactions suggest that although each of the mechanisms described here contributes to defence against fungi, there is no single essential mechanism but an interplay of multiple defences in the plant responding to attacks and counterattacks by the fungus. What has resulted is a multitude of defensive molecules to keep biochemists and molecular biologists busy and interested for years to come.

References
Epple P, Apel K and Bohlmann H (1997) Overexpression of an endogenous thionin enhances resistance of Arabidopsis against Fusarium oxysporum. The Plant Cell 9: 509520. Sandrock RW and VanEtten HD (1998) Fungal sensitivity to and enzymatic degradation of the phytoanticipin a-tomatine. Phytopathology 88: 137143. Skern T, Zorn M, Blaas D and Kuechler E (1990) Protease or protease inhibitor? Nature 344: 26. Woloshuk CP, Meulenho JS, Sela-Buurlage M, van den Elzen PJM and Cornelissen BJC (1991) Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. The Plant Cell 3: 619628.

Further Reading

Conclusions
Evolution has seen to it that plants are resistant to most pathogens, but because of the variety of pathogens,

Hartleb H, Heitefuss R and Hoppe H-H (eds) (1997) Resistance of Crop Plants Against Fungi. Jena: Gustav Fischer-Verlag. Yun D-J, Bressan RA and Hasegawa PM (1997) Plant antifungal proteins. Plant Breeding Reviews 14: 3988.

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