Visualizing plantpathogen interactions involving phenolics with histochemical stains

As most phenols are found as esters or glycosides, knowledge of their location in a cell or tissue is essential. It is typical that such phenols are sequestered or stored in the cell vacuole. This is important since all phenols are weak acids (see Chapter 2) and as such they are relatively toxic even to the cells in which they are synthesized. The toxicity of the compounds is significantly reduced when they are present as either esters or glycosides. The enzymes that cleave them are sometimes specific esterases and glycosidases but nonspecific enzymes may also cleave them. The enzymes are also located within the cell vacuole and in some cases have been shown to be present within the cell wall. Under environmental stresses, such as heat, cold and high light intensity, appropriate enzymes can be produced resulting in compound breakdown. The most typical situation, however, is that of disease where an invading pathogen such as a fungus perturbs cells or tissues causing them to respond by the synthesis of enzymes. It is also not uncommon for a single cell of the host to respond to the presence of an invading pathogen, a condition often referred to as a hypersensitive response. Although the hypersensitive response is a restricted phenomenon with the involvement of a very limited number of host cells, there is substantial evidence that host cells that surround the site of attempted infection are themselves stimulated or activated to a condition that allows for a much more rapid response if they themselves are perturbed by a pathogen that is attempting to infect the cell or tissue. The best-documented examples of this phenomenon are found in the barley powdery mildew disease interaction. The powdery mildew pathogen is Erysiphe graminis (Blumeria graminis), which is an obligate pathogen. It was shown that when a conidium of a compatible pathogen attempts to penetrate into a susceptible host, the penetration attempt is successful. However, if a conidium of an incompatible pathogen attempts to penetrate, the host cell responds in an incompatible manner and expresses resistance (Shiraishi et al., 1995).

Papillae are structures made by the plant in an attempt to contain and eliminate a pathogenic fungus. Papilla formation in maize leaves inoculated with Colletotrichum graminicola was reported by Politis and Wheeler (1973). These authors reported that even 'massive' papillae were unable to prevent penetration by the fungus. Their data were, however, not based on a time-sequence study of the events leading up to and including penetration and they did not present data on the number of successful penetrations. Papilla formation was also reported in isolated maize root cap cells after inoculation with C. graminicola (Sherwood, 1985). Differences in the frequency of papilla formation and penetration by the fungus were found among different maize lines.

Inoculation of maize roots with the non-pathogen Phytophthora cinnamomi also resulted in production of papillae (Hinch and Clarke, 1980). Callose was reported as the major component of the papillae, and carbohydrates but not proteins were identified. Lignin did not appear to be

Figure 4-7 (left). Characteristics of papilla formation by the epidermis of maize mesocotyls in response to attempted penetration by Colletotrichum graminicola and Helminthosporium maydis. a) Papilla formed by inbred B73Ht in response to C. graminicola, b) granulation (arrow) in B73Ht in response to H. maydis, c) granulation (arrow) and papilla formation in a single epidermal cell of the inbred B73Htrhm in response to H. maydis. All specimens stained with Toluidine Blue-O. Bars represent 10 ^M. Reprinted from Physiol. Mol. Plant Pathol. Vol. 31, Cadena-Gomez, G., and Nicholson, R.L., Papilla formation and associated peroxidase activity: A non-specific response to attempted fungal penetration of maize, pages 51-67, 1987, with permission from Elsevier.

Figure 4-7 (left). Characteristics of papilla formation by the epidermis of maize mesocotyls in response to attempted penetration by Colletotrichum graminicola and Helminthosporium maydis. a) Papilla formed by inbred B73Ht in response to C. graminicola, b) granulation (arrow) in B73Ht in response to H. maydis, c) granulation (arrow) and papilla formation in a single epidermal cell of the inbred B73Htrhm in response to H. maydis. All specimens stained with Toluidine Blue-O. Bars represent 10 ^M. Reprinted from Physiol. Mol. Plant Pathol. Vol. 31, Cadena-Gomez, G., and Nicholson, R.L., Papilla formation and associated peroxidase activity: A non-specific response to attempted fungal penetration of maize, pages 51-67, 1987, with permission from Elsevier.

Figure 4-8 (right). Histochemical reactions of maize (B73Htrhm) papillae. a) Specimen stained with Toluidine Blue-O. Blue-green color of the papilla and associated host cell wall indicate the presence of lignin. b) Specimen stained with phloroglucinol-HCl. Red to red-brown color indicates the presence of cinnamaldehyde end groups in lignin. c) Specimen stained for the presence of peroxidase by the syringaldazine procedure. Red color indicates the presence of peroxidase. Specimens were taken between 12 and 24 h after inoculation. Reprinted from Physiol. Mol. Plant Pathol. Vol. 31, Cadena-Gomez, G., and Nicholson, R.L., Papilla formation and associated peroxidase activity: A non-specific response to attempted fungal penetration of maize, pages 51-67, 1987, with permission from Elsevier.

present. The authors reported that papillae formed after penetration of roots may represent a wound response that allows repair of damaged cells or provides a barrier to toxic products produced by the fungus.

In maize a variety of stain procedures were used with light microscopy to demonstrate that papillae formed in response to attempted infection by Colletotrichum graminicola and Helminthosporium maydis are composed of lignin (Cadena-Gomez and Nicholson, 1987; Figures 4-7 and 4-8).

Different histochemical tests have been used for peroxidase identification. Benzidine (4.33) has been used as a staining reagent, as well as guaiacol (4.34) and pyrogallol (4.35). However, until the 1970s, no reliable methods were known that allowed a sharp discrimination between oxidase and peroxidase activities (Maehly and Chance, 1954). Harkin and Obst (1973) reported the syringaldazine (4.36) histochemical test for peroxidase. This test permitted the proof of exclusive peroxidase participation in the lignification process.

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Cytochemical identification of peroxidase was conducted in infected wound margins in the cell walls and degenerating cytoplasm of wheat leaves with 3,3'-diaminobenzidine (Thorpe and Hall, 1984). The same substrate was used to demonstrate peroxidase in the epidermis of wheat roots (Smith and O'Brian, 1979), and in roots and hypocotyls of cotton seedlings (Mueller and Beckman, 1978), and in bean leaves infected by the rust fungus Uromyces apendiculatus (Deising et al., 1992).

Peroxidase activities were also shown to occur during the bean rust infection process (Mendgen, 1975). In reed canary grass (Phalaris arundinacea L.), peroxidase was identified with the pyrogallol test (Vance and Sherwood, 1976).

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