21st Century Guidebook to Fungi, SECOND EDITION, by David Moore, Geoffrey D. Robson and Anthony P. J. Trinci

Table of Contents

1. Chapter 13: Ecosystem mycology: saprotrophs, and mutualisms between plants and fungi
2. Chapter 14: Fungi as pathogens of plants
   1. Fungal diseases and loss of world agricultural production
   2. A few examples of headline crop diseases
   3. The Rice Blast fungus Magnaporthe grisea (Ascomycota)
   4. Armillaria (Basidiomycota)
   5. Pathogens that produce haustoria (Ascomycota and Basidiomycota)
   6. Cercospora (Ascomycota)
   7. Ophiostoma (Ceratocystis) novo-ulmi (Dutch Elm disease or DED) (Ascomycota)
   8. Black stem rust (Puccinia graminis f. sp. tritici) threatens global wheat harvest
   9. Plant disease basics: the disease triangle
   10. Necrotrophic and biotrophic pathogens of plants
   11. The effects of pathogens on their hosts
   12. How pathogens attack plants
   13. Host penetration through stomatal openings
   14. Direct penetration of the host cell wall
   15. Enzymatic penetration of the host
   16. Pre-formed and induced defence mechanisms in plants
   17. Genetic variation in pathogens and their hosts: co-evolution of disease systems
   18. Chapter 14 References and further reading
   19. Buy a PDF of Chapter 14
3. Chapter 15: Fungi as symbionts and predators of animals

14.15 Enzymatic penetration of the host

Secreted proteins are common components of the descriptions above. Pathogenic fungi share a common lifestyle that involves penetration of the living plant; for biotrophs this also means penetrating plant cell walls, potentially suppression of the defences of the plant, and intimate contact with living host plant cell membranes as a means of acquiring nutrients. All of this requires investment of fungal gene products in the pathogenesis, and we have just referred to the large number of fungal genes that can be shown to be devoted to fungal pathogenicity in the hemibiotroph Magnaporthe oryzae. However, comparative analysis of genomes shows that the gene complement for toxin biosynthetic pathways and cell wall degrading enzymes of necrotrophic fungal pathogens, which kill and consume host cells, is even greater than that found in biotrophs.

As we have seen above, pathogenic fungi generally secrete a blend of hydrolytic enzymes, which includes cutinases, cellulases, pectinases, and proteinases, during germination and host penetration. Such enzymes are also produced by saprotrophs but their activities, substrate specificities and regulation are adapted to the needs of the pathogens and the nature of their host. For example, Alternaria brassicicola (Ascomycota), which causes black or dark leaf spot on many brassicas but is also found as a common saprotroph in soil, decaying plant material, wood, and on foods, produces different cutinase isozymes during saprotrophic and pathogenic stages (Knogge, 1998).

Penetrating a host leaf surface composed of cutin covered with wax is the first challenge to plant pathogens. Infection requires intricate sensing and signalling in both plant and pathogen (Knogge, 1998; van Kan, 2006). The pathogen must choose whether, when and where to germinate, develop an infection structure and/or produce enzymes and metabolites. It does this by sensing the physical and chemical environment, recognising hydrophobic and hydrophilic surfaces, as well as surface hardness and the chemical environment, too (Knogge, 1998; van Kan, 2006).

The grey mould pathogen Botrytis cinerea (Ascomycota) is a well-studied necrotroph that forms an appressorium that is probably not capable of penetrating the host by direct physical pressure. It lacks the highly melanised wall and the septum separating the appressorium from the germ tube, which are characteristic features of appressoria that generate high physical pressures like those of Blumeria and Magnaporthe. For B. cinerea appressoria, secreted enzymes, particularly cutinases and lipases, are the key to breaching the plant surface; the genome of Botrytis cinerea contains at least five cutinase genes and over a dozen lipase genes.

The tip of the penetration peg that emerges from the appressorium also generates H₂O₂ as a co-substrate for oxidases that degrade cuticular components and aid penetration. Early invasion of epidermal cells also involves pectinases (endopolygalacturonase). A common feature of plants that are most susceptible to B. cinerea is a relatively high content of pectin in the cell wall. This host preference of B. cinerea reflects an efficient pectinolytic machine comprising at least six endopolygalacturonase genes that are differentially regulated according to the nature of the host cell wall.

B. cinerea hastens host cell death in several ways, producing low molecular weight phytotoxic metabolites, the best-studied of which is the sesquiterpene botrydial (Fig. 9A).

Fig. 9. Phytotoxic metabolites of Botrytis cinerea. A, the sesquiterpene botrydial. B, the polyketide called botcinic acid.

Some strains of B. cinerea rely on botrydial alone to kill their host, while others produce additional toxins, such as botcinic acid (Fig. 9B).  B. cinerea, and the other common necrotrophic fungus Sclerotinia sclerotiorum (Ascomycota), also produce the dicarboxylic acid oxalate (Plassard & Fransson, 2009). Oxalate-deficient mutants are non-pathogenic but reversion to oxalate-production restores pathogenicity. Oxalic acid seems to serve several functions:

• it is a strong acid and several enzymes secreted by B. cinerea are active in an acidic environment (pectinases, proteinases and laccases).
• oxalate chelates divalent metal ions, particularly calcium and copper; most calcium in plant cells is stored in the cell wall, embedded in pectin. Oxalate extracts calcium ions from the pectin after partial hydrolysis by pectinases, and removal of calcium makes pectin more accessible to pectinases for further degradation.
• oxalate can reduce plant defence responses and trigger programmed cell death in plants (van Kan, 2006).

Host-specialised species of Botrytis, for example B. fabae and B. elliptica, produce phytotoxic proteins, known as host-selective toxins. These are necrosis and ethylene inducing proteins (NEPs), a family of proteins that induce plant plant cell death that were originally isolated from culture filtrates of Fusarium oxysporum but have since been found in a range of plant pathogenic microorganisms, including bacteria, Phytophthora spp., and fungi. Fungal extracellular enzymes are especially important in diseases known as soft rots (caused by several bacteria and fungi), to which fruit and some vegetables are prone. The enzymes concerned are pectinases, hemicellulases, cellulases, and ligninases (enzymes already discussed in the Chapter 10 Fungi in ecosystems; CLICK HERE to view the page). In commerce these are most important in relation to potential spoilage of fresh fruits and vegetables in the field, in transit or on displays; there are a few examples indicating that mycotoxins might have a role in the safety of fresh fruit at the retail level (Moss, 2008).

The general pattern coming out of these descriptions is that an infection by Botrytis species (and probably other heterotrophs) produces a range of toxic compounds of fungal origin that act as inducers of programmed cell death in the plant cell; in other words, host cell death requires the active participation of the pathogen and the host. Infection induces what is known as an oxidative burst. The oxidative burst is an early response to pathogen attack (in plant and animal cells) leading to the production of reactive oxygen species (ROS or superoxide) including hydrogen peroxide.

The major mechanisms involve either (plant) membrane-bound NADPH oxidases or peroxidases that can occur singly or in combination in different plant species. Most cells produce and detoxify ROS, as they form under normal conditions as by-products of successive one-electron reductions of molecular oxygen. Most cells also have protective mechanisms to maintain the lowest possible intracellular levels of ROS. When the cell is under stress, protective mechanisms are overridden by the rapid production of huge amounts of ROS, and this is ‘the oxidative burst’ (a similar reaction process has been known for many years in animal cells, particularly mammalian phagocytes, and is called the ‘respiratory burst’). However, in plants the phenomenon serves:

• to produce potential protectants against an invading pathogen: the oxidants themselves are directly protective, they function as substrates for oxidative cross-linking of the plant cell wall, and serve as diffusible signals to induce cell-protection genes in surrounding cells;
• as a central component of a highly amplified and integrated signal system, also involving salicylic acid and changes in cytoplasmic Ca²⁺, which underlies the expression the hypersensitive response that confers resistance to biotrophic pathogens. This is a programmed cell death involving nuclear condensation and expression of a panel of specific genes and activity of particular proteins. Characteristically, among these are metacaspases, which are Arg/Lys-specific equivalents of the Asp-specific caspases that contribute to apoptosis in animals. Metacaspases induce programmed cell death in plants and fungi.

The hypersensitive response protects the plant against biotrophic pathogens by rapidly killing the infected plant cell; so denying it to the invading pathogen before the fungus has had the opportunity to establish the feeding structures it needs to support the germinating spore that has initiated the infection. Sacrifice of one or a few cells of the leaf deprives the pathogens of a supply of food and effectively kills that germinating spore because an obligate biotroph has no saprotrophic ability. Necrotrophic pathogens, such as Botrytis cinerea and Sclerotinia sclerotiorum, however, can utilise dead tissue.

Indeed, it might be said that the goal of a necrotrophic plant pathogen is to kill its host cell in order to decompose plant biomass and convert it into fungal mass, so these fungi exploit a host defence mechanism for their pathogenicity (Govrin & Levine, 2000). Botrytis cinerea triggers an oxidative burst during cuticle penetration, using the oxidants to assist penetration, but the infection results in massive accumulation of hydrogen peroxide in the plant plasma membrane, which induces the hypersensitive response, killing the plant cells and thereby providing plant biomass for the fungus to consume saprotrophically (Knogge, 1998; van Kan, 2006). For this final stage the fungus can produce a full range of digestive enzymes, and a complete expression of digestive metabolism so that the fungus can take full advantage of the plant cell as a nutrient source (Jobic et al., 2007).

Updated July, 2019