P~ APT
E, SPECIFIC FUNGAL PROTEINS
Because the powdery mildew fungi are obligate parasites, they have not been easily amenable to physiological study. Nevertheless, progress has been made over the last
few years. Enzyme activity staining of isoelectric focusing protein electrophoresis gels (zymograms) has been used to assess the level of genetic variation among Bgh isolates (Koch and Kohler, 1990, 1991). These studies demonstrated the presence of the following enzymes in conidia: malate dehydrogenase, phosphogluconate dehydrogenase, glucose- 6-phosphate dehydrogenase, NADH-diaphorase, phosphoglucomutase, esterase, acid phosphatase, triose phosphate isomerase, glucose phosphate isomerase and superoxide dismutase. One of the first events to occur upon contact of a conidium with the surface of the barley leaf (or cellophane) is a release of liquid. Contact of this liquid with the leaf surface results in the disappearance of wax crystals from the surface (Kunoh et aI., 1988b) and erosion of the cuticle (Staub et aI., 1974; Nicholson et aI., 1988; Kunoh et aI., 1990). Esterase was released into the exudate within 15 to 30 minutes of contact (Nicholson et aI., 1988), and this second release could be inhibited by the translational inhibitor, cycloheximide (Kunoh et aI., 1990). The presence of esterase in this exudate suggests that the esterase activity observed is perhaps due to cutinase; the presence of cutinase in the infection exudate was confirmed using a specific assay by Pascholati et al. (1992). However, this latter study did not investigate the timing of release of the cutinase or its relationship to the individual esterase isozymes observed previously (Nicholson et aI., 1988). (See also Francis et aI., 1996.) Takahashi et al. (1985) used histological enzyme assays to look for hydrolytic enzymes associated with individual infection structures, and found esterase activity associated with primary and secondary germ tubes and their respective papillae. In this study, it was not possible to determine whether the enzymes were of host or pathogen origin.
Molecular studies of the powdery mildew fungus are still in their infancy. Only five gene sequences are published: two retroposon elements (Rasmussen et aI., 1993; Wei et aI., 1996), B-tubulin (Sherwood and Somerville, 1990) and two as yet unidentified gene transcripts present in germinating conidia (Justesen et aI., 1995).
IV. Resistance Mechanisms
A. PAPILLA FORMATION
In response to the PGT and to the appressorial lobes, the leaf forms papillae in an attempt to stop penetration. Papilla formation begins with the appearance of a cytoplasmic aggregate subjacent to these germ tubes.
1. Cytoplasmic Aggregate
In a normal epidermal cell, only a very thin layer of cytoplasm is found between the vacuolar tonoplast and the plasma membrane. The cytoplasm granulates locally in response to the fungal hyphae. In the case of the first appressorial lobe this happens at approximately 11 hours after inoculation and is completed within 5 to 10 min. (Bushnell and Zeyen, 1976; Aist and Israel, 1977). About half an hour later, the appressorium forms the penetration peg. Initiation of the penetration peg generally precedes initiation of the papilla by up to 1 hour, but it is not unusual that initiation of the papilla precedes initiation of the penetration peg (Aist and Israel, 1977). The cytoplasmic
88 H. Thordal-Christensen. P.L. Gregersen & D.B. Collinge
aggregate is characterized by high secretory activity where proteins, carbohydrates and phenols (Kita et aI., 1980; Russo and Bushnell, 1989) are exported across the host cell membrane in order to be built into the papilla. See also Akutsu et al. (1980).
A recent study, using inhibitors of actin and tubulin polymerization has demonstrated that re-organisation of microfilaments and microtubules is associated with the cytoplasmic aggregation and subsequent papilla formation (Kobayashi et al., 1997).
2. The Papilla
The papilla is a cell wall apposition located subjacent to the fungal germ tubes on the inner surface of the outer epidermal cell wall. Papillae subjacent to the PGTs are approximately 3 ~m in diameter while those formed subjacent to the appressoria are 4-5 ~m in diameter.
Papillae are surrounded by "haloes" which are 10-20 ~m in diameter (e.g. Thordal- Christensen and Smedegaard-Petersen, 1988b). A number of compounds have been demonstrated in the papillae and haloes. These include callose, phenolics, proteins, silicon and hydrogen peroxide (Kunoh and Ishizaki, 1976; Kita et aI., 1980; Zeyen et aI., 1983; Smart et aI., 1986; Russo and Bushnell, 1989, Thordal-Christensen et aI., 1997). A "basic staining material" observed by e.g. Russo and Bushnell (1989) is potentially a guanidine radical (Wei et aI., 1994). The phenolic compounds in the papillae and haloes have been detected by UV -fluorescence, lacmoid, methylene blue, resorcinol and toluidine blue staining; nevertheless, the phenolic substances remain uncharacterized in detail. Several attempts have failed to demonstrate the presence of lignin using phloroglucinol-HCl (e.g.
Smart et aI., 1986; Russo and Bushnell, 1989; Wei et al., 1994). Lignin can, on the other hand, be demonstrated in wheat papilla using this stain (e.g. Wei et aI., 1994). Specific proteins in the papilla include hydrolytic enzymes (Takahashi et al., 1985), leaf thionins (Ebrahim- Nesbat et aI., 1993) and peroxidase (Scott-Craig et al., 1995). It has been demonstrated that proteins are covalently cross-bound in the papilla structure (Thordal-Christensen et aI., 1997), and the immobility of e.g. the phenolics suggests that they are covalently cross-bound as well. The papilla has a complex ultrastructure. Heitefuss and co-workers have defined several types of papilla with different organizations of e.g. layers and more or less irregular structures (see Heitefuss and Ebrahim-Nesbat, 1986). See also Hippe-Sanwald et al. (1992).
3. Penetration Success
It is generally accepted that the resistance manifested at the early penetration stage is largely race-non-specific (see chapter by Bert Keller et al.) and is superimposed on resistance conferred by specific resistance genes, which is manifested through the hypersensitive response (see below). Nevertheless, the outcome of the "battle" between the penetration hypha and the papilla is dependent on a number of variable factors.
a. Genotypes. The ml-o resistance results in earlier and larger papillae (Skou et aI., 1984; Gold et al., 1986) which efficiently stop the penetration attempt. It remains an open question as to which specific component makes these papillae so efficient (see below). However, Bgh isolates with a higher infection rate on ml-o resistant plants have been identified (Schwarzbach, 1979; Lyngkjrer et aI., 1995). Data obtained by Carver (1986) in a study of varieties with different level of partial resistance suggest that the host's genetic background is important for papilla resistance.
b. PGT-Activated Defense. As discussed above, the PGT activates defense reactions which are manifested a few hours later against the penetration from appressoria.
Resistance induced within 16 hours of exposure to Bgh, during which period the PGT is a major contributor to defense activation, is manifested as reduced penetration success by Bgh. This reduction correlates with an increased papilla diameter (Thordal- Christensen and Smedegaard-Petersen, 1988b).
c. Host Cell Type. There is a pronounced correlation between the size/location of the epidermal cells and their penetration. The short cells near the stomata, which are placed in rows, are more readily penetrated than the long cells between the rows of stomata (e.g. Johnson et aI., 1979; Thordal-Christensen and Smedegaard-Petersen, 1988b; Koga et aI., 1990; Gorg et aI., 1993). A possible explanation for this might be that long cells are more likely to be encountered by PGTs, and this induces resistance to later penetration attempts. The very small stomatal subsidiary cells appear to exhibit inefficient papilla resistance, and they are susceptible even in ml-o resistant plants (J0rgensen and Mortensen, 1977).
4. How Do Papillae Work?
Many questions in relation to papilla-based resistance remain unanswered. For instance, where and how is the penetration peg arrested? A few published micrographs suggest that the peg stops inside the papilla (e.g. Edwards and Allen, 1970; Heitefuss and Ebrahim-Nesbat, 1986; Aist et aI., 1988). This is perhaps to be expected; however, the matter needs clarification. Why does the penetration peg stop growing - i.e. which papilla component is significant? Use of specific enzyme inhibitors has contributed information to this question.
Callose is a major papilla constituent and it has been suggested to be necessary for papilla efficacy, particularly in ml-o resistant plants (Skou et aI., 1984; Bayles et aI., 1990).
Callose is synthesized from UDP-glucose by the plasma membrane bound callose synthase. In vitro callose synthase activity, measured in the microsomal fraction of barley leaf epidermal tissue, has been shown to be independent of the ml-o resistance gene as well as of inoculation (Pedersen, 1992), i.e. the enzyme is constitutive and its activity does not depend on resistance genotype or inoculation. The constitutive expression suggests that a regulatory mechanism for this enzyme exists. Plant callose synthase is inactive when the concentration of free Ca2+ in the cytoplasm remains around 0.1 ~, while half-max. activity occurs when this concentrations is around 0.8 ~ (Kauss, 1992). Indeed, there is also evidence which suggests that papilla formation in ml-o resistant plants is Ca2+-dependent (Gold et aI., 1986), and that inhibition of callose formation using a substrate analogue (2-deoxy-D-glucose) increases the penetration rate of ml-o resistant and, but to a lesser extent, in susceptible plants (Bayles et aI., 1990).
Phenylalanine ammonia lyase (PAL) activity increases following inoculation with Bgh (see below) contributing, presumably, precursors for the synthesis of phenolics.
The significance of these phenolic compounds has been studied by Carver and co-workers in barley, wheat and oat. Generally, the use of specific inhibitors towards PAL and cinnamyl alcohol dehydrogenase (CAD) led to a reduction in papilla autofluorescence
90 H. Thordal-Christensen, P.L. Gregersen & D.H. Collinge
and at the same time increased the penetration rate (Carver et ai., 1992, 1994, 1996).
CAD is a key enzyme for production of lignin precursors, so these data suggest that lignin or lignin-like compounds are present and important in barley papilla in spite of their inability to be detected histochemically (see above).
It appears that different components of papillae contribute to the efficacy of this cell wall apposition in preventing penetration and successful colonization.